Cyanoisoquinoline compounds and methods of use thereof

ABSTRACT

The present invention relates to cyanoisoquinoline compounds suitable for use in treating hypoxia inducible factor-mediated and/or erythropoietin-associated conditions. The cyanoisoquinoline compounds of the invention have the following structure:

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit under 35 U.S.C. §119 (e) ofU.S. Provisional Application Ser. No. 60/762,780, filed Jan. 27, 2006,which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compounds capable ofdecreasing hypoxia inducible factor (HIF) hydroxylase enzyme activity,thereby increasing the stability and/or activity of HIF. In particular,the compounds increase endogenous erythropoietin, ex vivo and in vivo.

2. State of the Art

Hypoxia inducible factor (HIF) is a basic helix-loop-helix (bHLH) PAS(Per/Arnt/Sim) transcriptional activator that mediates changes in geneexpression in response to changes in cellular oxygen concentration. HIFis a heterodimer containing an oxygen-regulated α-subunit (HIFα), and aconstitutively expressed β-subunit (HIFβ), also known as arylhydrocarbon receptor nuclear transporter (ARNT). In oxygenated(normoxic) cells, HIFα subunits are rapidly degraded by a mechanism thatinvolves ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL)E3 ligase complex. Under hypoxic conditions, HIFα is not degraded, andan active HIFα/β complex accumulates in the nucleus, and activates theexpression of several genes including glycolytic enzymes, glucosetransporters, erythropoietin (EPO), and vascular endothelial growthfactor (VEGF). (Jiang, et al., (1996) J. Biol. Chem., 271:17771-17778;Iliopoulus, et al., (1996) Proc. Natl. Acad. Sci. USA, 93:10595-10599;Maxwell, et al., (1999), Nature, 399:271-275; Sutter, et al., (2000)Proc. Natl. Acad. Sci. USA, 97:4748-4753; Cockman, et al., (2000) J.Biol. Chem., 275:25733-25741; and Tanimoto, et al., (2000) EMBO. J.19:4298-4309.)

Levels of HIFα are elevated in most cells in response to hypoxia, andHIFα is induced in vivo when animals are subjected to anemia or hypoxia.HIFα levels rise within a few hours after the onset of hypoxia, andinduce numerous beneficial cellular processes including cytoprotectiveeffects, enhanced erythropoiesis, and physiological adaptation toischemic or hypoxic states. Induction of HIFα is potentially beneficialin conditions such as myocardial acute ischemia and early infarction,pulmonary hypertension, inflammation, and anemia.

HIFα levels are also increased by a number of factors that mimichypoxia, including iron chelators such as desferrioxamine (DFO), anddivalent metal salts such as CoCl₂. Additionally, compounds originallyidentified as inhibitors of procollagen prolyl hydroxylase enzymes havebeen found to stabilize HIFα. Examples of such compounds can be found,e.g., in Majamaa et al. (1984) Eur J Biochem 138:239-245; Majamaa et al.(1985) Biochem J 229:127-133; Kivirikko and Myllyharju (1998) MatrixBiol 16:357-368; Bickel et al. (1998) Hepatology 28:404-411; Friedman etal. (2000) Proc Natl Acad Sci USA 97:4736-4741; Franklin (1991) BiochemSoc Trans 19:812-815; and Franklin et al. (2001) Biochem J 353:333-338.Additionally, compounds that stabilize HIFα have been described in,e.g., International Publication Nos. WO 03/049686, WO 02/074981, WO03/080566, WO 2004/108681, and WO 2006/094292.

There remains a need for compounds that are effective in the preventionof disorders associated with HIF, including anemia, and tissue damagecaused by ischemia that occurs due to, e.g., atherosclerosis, diabetes,and pulmonary disorders such as pulmonary embolism, and the like. Thus,compounds that modulate HIF, which can be used to treat, and preventHIF-associated disorders including conditions involving anemia,ischemia, and hypoxia, are provided herein.

SUMMARY OF THE INVENTION

The present invention is directed to novel compounds and methods ofusing the compounds to modulate hydroxylation of HIFα and/or increaseendogenous erythropoietin (EPO). In particular, this invention isdirected to isoquinoline compounds with a cyano group at the C-1position, which compounds enhance the production of endogenous EPO (see,e.g., Table 1).

In one aspect, the invention provides compounds of formula I:

wherein:

R is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl;

R¹, R², R³ and R⁴ are independently selected from the group consistingof hydrogen, halo, cyano, hydroxyl, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, —OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷ is selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and

R⁵ and R⁶ are independently selected from the group consisting ofhydrogen or C₁₋₃ alkyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In one embodiment, the invention is directed to compounds of formula Ia:

wherein:

q is 0 or 1;

R²¹, R²², R²³ and R²⁴ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, alkyl, substituted alkyl,haloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy,substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy,substituted heteroaryloxy, heterocyclyl, substituted heterocyclyl,heterocyclyloxy, amino, and substituted amino wherein at least two ofR²¹, R²², R²³ and R²⁴ are hydrogen; and

R²⁵ is selected from hydrogen or methyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In another aspect, the invention provides compounds of formula II:

wherein:

R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, halo, hydroxyl, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, —OR³⁷, —SR³⁷, —SOR³⁷, and —SO₂R³⁷ wherein R³⁷ is selected fromthe group consisting of alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl; and

R³⁵ is hydrogen or methyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In one embodiment, the invention is directed to compounds of formula II,wherein

R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, alkyl, substituted alkyl,haloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy,substituted aryloxy, heteroaryloxy, substituted heteroaryloxy,cycloaryloxy, substituted cycloaryloxy, amino, and substituted amino;and

R³⁵ is hydrogen or methyl;

-   -   or pharmaceutically acceptable salts, tautomers, stereoisomers,        solvates, and/or prodrugs thereof.

In some embodiments, the invention provides compounds of formula IIwherein at least three of R³¹, R³², R³³, and R³⁴ are hydrogen.

The invention also provides pharmaceutical compositions comprising oneor more compounds of formula I, and/or II, and a pharmaceuticallyacceptable excipient. In some embodiments, the composition furthercomprises at least one additional therapeutic agent. In someembodiments, the agent is selected from the group consisting of vitaminB12, ferrous sulfate, folic acid, and/or erythropoietin or anerythropoiesis stimulating protein (ESP).

The invention is also directed to methods of inhibiting the activity ofat least one HIF hydroxylase enzyme, the method comprising bringing intocontact the HIF hydroxylase enzyme and an inhibitory-effective amount ofa compound of the invention. In one embodiment, the HIF hydroxylaseenzyme is an asparaginyl hydroxylase such as Factor Inhibiting HIF(FIH). In another embodiment, the HIF hydroxylase enzyme is a prolylhydroxylase including, but not limited to, a HIF prolyl hydroxylaseselected from the group consisting of human EGLN1, EGLN2, or EGLN3, oran orthologous enzyme from another species.

The invention is also directed to methods of treating, pretreating, ordelaying onset of a condition associated with or mediated at least inpart by hypoxia inducible factor (HIF), the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I and/or II or a pharmaceutical composition producedtherefrom. In one embodiment, the condition associated with or mediatedby HIF is tissue damage associated with ischemia or hypoxia. In oneaspect, the ischemia is an acute ischemic event including, but notlimited to, an acute ischemic event selected from the group consistingof myocardial infarction, pulmonary embolism, intestinal infarction,ischemic stroke, and renal ischemic-reperfusion injury. In anotheraspect, the ischemia is a chronic ischemic event including, but notlimited to, a chronic ischemic event selected from the group consistingof cardiac cirrhosis, macular degeneration, chronic kidney failure, andcongestive heart failure.

The invention is also directed to methods of treating, pretreating, ordelaying onset of a condition associated with or mediated at least inpart by erythropoietin (EPO), the method comprising administering to apatient a therapeutically effective amount of a compound of formula Iand/or II or a pharmaceutical composition produced therefrom.

The invention is also directed to methods of treating, pretreating, ordelaying onset of anemia, the method comprising administering to apatient a therapeutically effective amount of a compound of formula Iand/or II or a pharmaceutical composition produced therefrom.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, cell lines, assays, and reagents described, asthese may vary. It is also to be understood that the terminology usedherein is intended to describe particular embodiments of the presentinvention, and is in no way intended to limit the scope of the presentinvention as set forth in the appended claims.

A. Compounds of the Invention

The present invention provides compounds represented by formula I:

wherein:

R is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl;

R¹, R², R³ and R⁴ are independently selected from the group consistingof hydrogen, halo, cyano, hydroxyl, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, —OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷ is selected from thegroup consisting of alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and

R⁵ and R⁶ are independently selected from the group consisting ofhydrogen or C₁₋₃ alkyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In some embodiments, the invention provides compounds of formula Iwherein at least two of R¹, R², R³ and R⁴ are hydrogen.

In some embodiments, the invention provides compounds of formula Iwherein the nitrogen in the isoquinoline ring is N-oxide.

In some embodiments, R¹, R², R³, and R⁴ are independently selected fromthe group consisting of hydrogen, hydroxyl, halo, substituted alkylincluding haloalkyl and trifluoromethyl, aryl, —OR⁷, —SR⁷, and —SO₂R⁷wherein R⁷ is selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl. In particular embodiments, R¹, R², R³, and R⁴ are selectedfrom the group consisting of hydrogen, hydroxyl, phenyl, chloro,trifluoromethyl, benzyl, benzyloxy, methoxy, butoxy, isopropoxy,phenoxy, 4-fluorophenoxy, 2-methoxyphenoxy, 3-methoxyphenoxy,4-methoxyphenoxy, 2,6-dimethylphenoxy, 2-ethyl-6-methylphenoxy,2,4,6-trimethylphenoxy, 4-chloro-2,6-dimethylphenoxy, 4-propoxyphenoxy,2,3-dihydro-benzofuran-5-yloxy, 2-methyl-benzothiazol-6-yloxy,2-dimethylamino-benzooxazol-5-yloxy,2-morpholin-4-yl-benzothiazol-6-yloxy, 2-methyl-benzooxazol-6-yloxy,benzo[1,3]dioxo-5-yloxy, phenylsulfanyl, phenylsulfonyl, andcyclohexyloxy.

In some embodiments, R¹, R², R³, and R⁴ are selected from hydrogen,halo, haloalkyl, alkyl, alkoxy, aryloxy and substituted aryloxy. Inparticular embodiments, R¹, R², R³, and R⁴ are selected from hydrogen,chloro, methoxy, trifluoromethyl, phenoxy, and 4-fluorophenoxy.

In some embodiments, R¹ and R² are hydrogen. In other embodiments R³ andR⁴ are hydrogen. In other embodiments R² and R³ are hydrogen. In otherembodiments, R¹ and R⁴ are hydrogen. In other embodiments, R¹, R², R³,and R⁴ are hydrogen.

In still other embodiments, R², R³, and R⁴ are hydrogen. In particularembodiments wherein R², R³, and R⁴ are hydrogen, R¹ is phenyl, phenoxyor 4-fluorophenxoy.

In still other embodiments, R¹, R², and R⁴ are hydrogen. In someembodiments R³ is selected from the group consisting of hydroxyl, halo,haloalkyl, substituted alkyl, —OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷is selected from the group consisting of alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl. In particularembodiments, R³ is selected from the group consisting oftrifluoromethyl, chloro, hydroxyl, benzyl, methoxy, isopropoxy, butoxy,benzyloxy, phenoxy, 4-fluorophenoxy, 2,6-dimethylphenoxy,4-methoxyphenoxy, 2-dimethylamino-benzooxazol-5-yloxy,benzo[1,3]dioxo-5-yloxy, and phenylsulfanyl. In certain embodimentswherein R¹, R², and R⁴ are hydrogen, R³ is phenoxy, 4-fluorophenoxy,trifluoromethyl, or chloro.

In still other embodiments, R¹, R², and R³ are hydrogen. In someembodiments R⁴ is selected from the group consisting of phenyl, phenoxy,2-methoxyphenoxy, 3-methoxyphenoxy, 4-methoxyphenoxy, and4-fluorophenoxy. In certain embodiments wherein R¹, R², and R³ arehydrogen, R⁴ is phenoxy or 4-fluorophenoxy.

In some embodiments R¹, R³ and R⁴ are hydrogen. In some embodiments, R²is selected from the group consisting of halo, cyano, hydroxyl, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, —OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷wherein R⁷ is selected from the group consisting of alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In particular embodiments, R² isselected from the group consisting of halo, —OR⁷, —SR⁷, —SOR⁷, and—SO₂R⁷ wherein R⁷ is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl. In certain embodiments, R² is selected from thegroup consisting of chloro, methoxy, isopropoxy, phenoxy,4-fluorophenoxy, 4-methoxyphenoxy, 2,6-dimethylphenoxy,2-ethyl-6-methylphenoxy, 2,4,6-trimethylphenoxy,4-chloro-2,6-dimethylphenoxy, 4-propoxyphenoxy,2,3-dihydro-benzofuran-5-yloxy, 2-methyl-benzothiazol-6-yloxy,2-dimethylamino-benzooxazol-5-yloxy,2-morpholin-4-yl-benzothiazol-6-yloxy, 2-methyl-benzooxazol-6-yloxy,benzo[1,3]dioxo-5-yloxy, phenylsulfonyl, phenylsulfanyl, andcyclohexyloxy. In certain embodiments wherein, R¹, R³, and R⁴ arehydrogen, R² is methoxy, phenoxy, or 4-fluorophenoxy.

In some embodiments, R⁵ is hydrogen. In other embodiments, R⁵ is methyl.

In some embodiments, R⁶ is hydrogen. In other embodiments, R⁶ is methyl.

In some embodiments, R is hydrogen. In other embodiments, R is methyl.

In one aspect, the invention provides compounds of formula II:

wherein:

R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, cyano, hydroxyl, halo, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, —OR³⁷, —SR³⁷, —SOR³⁷, and —SO₂R³⁷ wherein R³⁷ isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl; and

R³⁵ is hydrogen or methyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In some embodiments, the invention provides compounds of formula IIwherein at least three of R³¹, R³², R³³, and R³⁴ are hydrogen.

In one embodiment, the invention is directed to compounds of formula II,wherein

R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, alkyl, substituted alkyl,haloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy,substituted aryloxy, heteroaryloxy, substituted heteroaryloxy,cyclalkoxy, substituted cycloalkoxy, amino, and substituted amino; and

R³⁵ is hydrogen or methyl;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

In another embodiment, the invention is directed to compounds of formulaII, wherein

R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, substituted alkyl, aryl, aryloxy, andsubstituted aryloxy; and

R³⁵ is hydrogen;

or pharmaceutically acceptable salts, tautomers, stereoisomers,solvates, and/or prodrugs thereof.

Compounds of the invention include, but are not limited to, thefollowing compounds:{[1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic acid,2-(S)-[(1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl]-amino}-aceticacid,2-(S)-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,2-(R)-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-7-(4-fluorophenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-(trifluoromethyl)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-chloro-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-6-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(7-benzyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[6-(4-chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(6-benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[7-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[6-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester,[(1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,(S)-2-[(1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,(R)-2-[(1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(6-chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(7-butoxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(7-benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid, and [(1-cyano-4,7-dihydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid.

It will be self-evident to the skilled person that many of theembodiments described above are not mutually exclusive, and may becombined to provide further specific embodiments of the invention. Suchspecific embodiments are explicitly envisaged herein. Similarly, thedependent claims may be made dependent on any preceding claim, providedthat the embodiments described are not mutually exclusive.

B. Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.(See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; D. M. Weir, and C. C.Blackwell, eds. (1986) Handbook of Experimental Immunology, Vols. I-IV(Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Vols. I-III,Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999)Short Protocols in Molecular Biology, 4^(th) edition, John Wiley & Sons;Ream et al., eds. (1998) Molecular Biology Techniques: An IntensiveLaboratory Course, Academic Press; Newton & Graham eds., 1997 PCR(Introduction to Biotechniques Series), 2nd ed. (Springer Verlag).

The term “anemia” as used herein refers to any abnormality in hemoglobinor erythrocytes that leads to reduced oxygen levels in the blood. Anemiacan be associated with abnormal production, processing, or performanceof erythrocytes and/or hemoglobin. The term anemia refers to anyreduction in the number of red blood cells and/or level of hemoglobin inblood relative to normal blood levels.

Anemia can arise due to conditions such as acute or chronic kidneydisease, infections, inflammation, cancer, irradiation, toxins,diabetes, and surgery. Infections may be due to, e.g., virus, bacteria,and/or parasites, etc. Inflammation may be due to infection orautoimmune disorders, such as rheumatoid arthritis, etc. Anemia can alsobe associated with blood loss due to, e.g., stomach ulcer, duodenalulcer, hemorrhoids, cancer of the stomach or large intestine, trauma,injury, surgical procedures, etc. Anemia is further associated withradiation therapy, chemotherapy, and kidney dialysis. Anemia is alsoassociated with HIV-infected patients undergoing treatment withazidothymidine (zidovudine) or other reverse transcriptase inhibitors,and can develop in cancer patients undergoing chemotherapy, e.g., withcyclic cisplatin- or non-cisplatin-containing chemotherapeutics.Aplastic anemia and myelodysplastic syndromes are diseases associatedwith bone marrow failure that result in decreased production oferythrocytes. Further, anemia can result from defective or abnormalhemoglobin or erythrocytes, such as in disorders including microcyticanemia, hypochromic anemia, etc. Anemia can result from disorders iniron transport, processing, and utilization, see, e.g., sideroblasticanemia, etc.

The terms “disorders,” “diseases,” and “conditions” are used inclusivelyand refer to any condition deviating from normal.

The terms “anemic conditions” and “anemic disorders” refer to anycondition, disease, or disorder associated with anemia. Such disordersinclude, but are not limited to, those disorders listed above. Anemicdisorders further include, but are not limited to, aplastic anemia,autoimmune hemolytic anemia, bone marrow transplantation, Churg-Strausssyndrome, Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome,graft versus host disease, hematopoietic stem cell transplantation,hemolytic uremic syndrome, myelodysplastic syndrome, nocturnalparoxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, purered-cell aplasia, purpura Schoenlein-Henoch, sideroblastic anemia,refractory anemia with excess of blasts, rheumatoid arthritis, Shwachmansyndrome, sickle cell disease, thalassemia major, thalassemia minor,thrombocytopenic purpura, etc.

The term “erythropoietin-associated conditions” is used inclusively andrefers to any condition associated with below normal, abnormal, orinappropriate modulation of erythropoietin. Erythropoietin-associatedconditions include any condition wherein an increase in EPO level wouldprovide therapeutic benefit. Levels of erythropoietin associated withsuch conditions can be determined by any measure accepted and utilizedby those of skill in the art. Erythropoietin-associated conditionsinclude anemic conditions such as those described above.

Erythropoietin-associated conditions further include neurologicaldisorders and/or injuries, including cases of stroke, trauma, epilepsy,neurodegenerative disease and the like, wherein erythropoietin mayprovide a neuroprotective effect. Neurodegenerative diseasescontemplated by the invention include Alzheimer's disease, Parkinson'sdisease, Huntington's disease, and the like.

The term “erythropoietin” refers to any recombinant or naturallyoccurring erythropoietin or ESP including, e.g., human erythropoietin(GenBank Accession No. AAA52400; Lin et al. (1985) Proc Nat'l Acad. Sci.USA 82:7580-7584), EPOETIN human recombinant erythropoietin (Amgen,Inc., Thousand Oaks Calif.), ARANESP human recombinant erythropoietin(Amgen), PROCRIT human recombinant erythropoietin (Ortho BiotechProducts, L.P., Raritan N.J.), glycosylated erythropoietin such as thosedescribed in U.S. Pat. No. 6,930,086 (which is incorporated byreference), etc.

The term “HIFα” refers to the alpha subunit of hypoxia inducible factorprotein. HIFα may be any human or other mammalian protein, or fragmentthereof, including human HIF-1α (Genbank Accession No. Q16665), HIF-2α(Genbank Accession No. AAB41495), and HIF-3α (Genbank Accession No.AAD22668); murine HIF-1α (Genbank Accession No. Q61221), HIF-2α (GenbankAccession No. BAA20130 and AAB41496), and HIF-3α (Genbank Accession No.AAC72734); rat HIF-1α (Genbank Accession No. CAA70701), HIF-2α (GenbankAccession No. CAB96612), and HIF-3α (Genbank Accession No. CAB96611);and bovine HIF-1α (Genbank Accession No. BAA78675). HIFα may also be anynon-mammalian protein or fragment thereof, including Xenopus laevisHIF-1α (Genbank Accession No. CAB96628), Drosophila melanogaster HIF-1α(Genbank Accession No. JC4851), and chicken HIF-1α (Genbank AccessionNo. BAA34234). HIFα gene sequences may also be obtained by routinecloning techniques, for example by using all or part of a HIFα genesequence described above as a probe to recover and determine thesequence of a HIFα gene in another species.

A fragment of HIFα includes any fragment retaining at least onefunctional or structural characteristic of HIFα. Fragments of HIFαinclude, e.g., the regions defined by human HIF-1α from amino acids 401to 603 (Huang et al., supra), amino acid 531 to 575 (Jiang et al. (1997)J. Biol. Chem. 272:19253-19260), amino acid 556 to 575 (Tanimoto et al.,supra), amino acid 557 to 571 (Srinivas et al. (1999) Biochem BiophysRes. Commun 260:557-561), and amino acid 556 to 575 (Ivan and Kaelin(2001) Science 292:464-468). Further, HIFα fragments include anyfragment containing at least one occurrence of the motif LXXLAP. (SeeGenbank Accession No. Q16665.)

The term “fragment” can refer to any portion of a sequence that retainsat least one structural or functional characteristic of the protein.Fragments are of any length but preferably are of about 5 to 100 aminoacids in length, particularly of about 15 to 50 amino acids in length,and more particularly of about 20 amino acids in length. Where “aminoacid sequence” is used to refer to the polypeptide sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative sequence associated with the recited protein molecule.

The term “related proteins” as used herein, for example, to refer toproteins related to HIFα prolyl hydroxylase, encompasses other2-oxoglutarate dioxygenase enzymes, especially those family members thatsimilarly require Fe²⁺, 2-oxoglutarate, and oxygen to maintainhydroxylase activity. Such enzymes include, but are not limited to,e.g., procollagen lysyl hydroxylase, procollagen prolyl 4-hydroxylase,and Factor Inhibiting HIF (FIH), an asparaginyl hydroxylase responsiblefor regulating transactivation of HIFα. (GenBank Accession No. AAL27308;Mahon et al. (2001) Genes Dev 15:2675-2686; Lando et al. (2002) Science295:858-861; and Lando et al. (2002) Genes Dev 16:1466-1471. See alsoElkins et al. (2002) J Biol Chem C200644200, etc.).

The term “HIF hydroxylase” refers to any enzyme that modifies the alphasubunit of HIF by hydroxylation of one or more amino acid residues. HIFhydroxylases include Factor Inhibiting HIF (FIH) (GenBank AccessionAAL27308; Mahon et al. (2001) Genes Dev 15:2675-2686; Lando et al.(2002) Science 295:858-861; and Lando et al. (2002) Genes Dev16:1466-1471, which modifies at least one asparagine residue foundwithin HIFα. (Also, see, Elkins et al. (2002) J Biol Chem C200644200.)HIF hydroxylases also include HIF prolyl hydroxylases (HIF PHs), whichmodify proline residues found within HIFα.

The terms “HIF prolyl hydroxylase” and “HIF PH” refer to any enzymecapable of hydroxylating a proline residue in the HIF protein.Preferably, the proline residue hydroxylated by HIF PH includes theproline found within the motif LXXLAP. HIF PH includes members of theEgl-Nine (EGLN) gene family described by Taylor (2001, Gene275:125-132), and characterized by Aravind and Koonin (2001, Genome Biol2: RESEARCH 0007), Epstein et al. (2001, Cell 107:43-54), and Bruick andMcKnight (2001, Science 294:1337-1340). Examples of HIF PH enzymesinclude human SM-20 (EGLN1) (GenBank Accession No. AAG33965; Dupuy etal. (2000) Genomics 69:348-54), EGLN2 isoform 1 (GenBank Accession No.CAC42510; Taylor, supra), EGLN2 isoform 2 (GenBank Accession No. NP060025), and EGLN3 (GenBank Accession No. CAC42511; Taylor, supra);mouse EGLN1 (GenBank Accession No. CAC42515), EGLN2 (GenBank AccessionNo. CAC42511), and EGLN3 (SM-20) (GenBank Accession No. CAC42517); andrat SM-20 (GenBank Accession No. AAA19321). Additionally, HIF PH mayinclude Caenorhabditis elegans EGL-9 (GenBank Accession No. AAD56365)and Drosophila melanogaster CG1114 gene product (GenBank Accession No.AAF52050). HIF PH also includes any portion of the foregoing full-lengthproteins that retain at least one structural or functionalcharacteristic.

The term “excipient” as used herein means an inert or inactive substanceused in the production of pharmaceutical products or other tablets,including without limitation any substance used as a binder,disintegrant, coating, compression/encapsulation aid, cream or lotion,lubricant, parenteral, sweetener or flavoring, suspending/gelling agent,or wet granulation agent. Binders include, e.g., carbopol, povidone,xanthan gum, etc.; coatings include, e.g., cellulose acetate phthalate,ethylcellulose, gellan gum, maltodextrin, etc.;compression/encapsulation aids include, e.g., calcium carbonate,dextrose, fructose dc, honey dc, lactose (anhydrate or monohydrate;optionally in combination with aspartame, cellulose, or microcrystallinecellulose), starch dc, sucrose, etc.; disintegrants include, e.g.,croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creamsand lotions include, e.g., maltodextrin, carrageenans, etc.; lubricantsinclude, e.g., magnesium stearate, stearic acid, sodium stearylfumarate, etc.; materials for chewable tablets include, e.g., dextrose,fructose dc, lactose (monohydrate, optionally in combination withaspartame or cellulose), etc.; parenterals include, e.g., mannitol,povidone, etc.; plasticizers include, e.g., dibutyl sebacate,polyvinylacetate phthalate, etc.; suspending/gelling agents include,e.g., carrageenan, sodium starch glycolate, xanthan gum, etc.;sweeteners include, e.g., aspartame, dextrose, fructose dc, sorbitol,sucrose dc, etc.; and wet granulation agents include, e.g., calciumcarbonate, maltodextrin, microcrystalline cellulose, etc.

The term “subject” is used herein in its broadest sense. Subjects mayinclude isolated cells, either prokaryotic or eukaryotic, or tissuesgrown in culture. In certain embodiments, a subject is an animal,particularly an animal selected from a mammalian species including rat,rabbit, bovine, ovine, porcine, canine, feline, murine, equine, andprimate, particularly human.

The term “alkyl” refers to monovalent hydrocarbyl groups having from 1to 10 carbon atoms, more particularly from 1 to 5 carbon atoms, and evenmore particularly 1 to 3 carbon atoms. This term is exemplified bygroups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl,n-pentyl and the like.

The term “substituted alkyl” refers to an alkyl group, of from 1 to 10carbon atoms, particularly 1 to 5 carbon atoms, having from 1 to 5substituents, more particularly 1 to 3 substituents, each of which isindependently selected from the group consisting of alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aryl, substituted aryl, aryloxy, substituted aryloxy, cyano (—CN),halogen, hydroxyl (—OH), nitro (—NO₂), oxo (═O), thioxo (═S), carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, thiol, alkylthio,substituted alkylthio, arylthio, substituted arylthio, cycloalkylthio,substituted cycloalkylthio, heteroarylthio, substituted heteroarylthio,heterocyclicthio, substituted heterocyclicthio, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —NR⁴⁰S(O)₂-alkyl,—NR⁴⁰S(O)₂-substituted alkyl, —NR⁴⁰S(O)₂-aryl, —NR⁴⁰S(O)₂-substitutedaryl, —NR⁴⁰S(O)₂-heteroaryl, —NR⁴⁰S(O)₂-substituted heteroaryl,—NR⁴⁰S(O)₂-heterocyclic, —NR⁴⁰S(O)₂-substituted heterocyclic,—OSO₂—NR⁴⁰R⁴⁰, —NR⁴⁰S(O)₂—NR⁴⁰-alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted alkyl,—NR⁴⁰S(O)₂—NR⁴⁰-aryl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted aryl,—NR⁴⁰S(O)₂—NR⁴⁰-heteroaryl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted heteroaryl,—NR⁴⁰S(O)₂—NR⁴⁰-heterocyclic, and —NR⁴⁰S(O)₂—NR⁴⁰-substitutedheterocyclic where each R⁴⁰ is independently selected from hydrogen oralkyl.

The term “haloalkyl” refers to an alkyl group substituted with from 1 to5, and particularly 1 to 3 halogen atoms. Preferably, the halogen atomis fluoro or chloro. Suitable haloalkyl moieties include, but are notlimited to, —CF₃, —CH₂CF₃.

The term “alkoxy” refers to the group “alkyl-O—” which includes, by wayof example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy and the like.

The term “substituted alkoxy” refers to the group “substitutedalkyl-O—”.

The term “acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)— provided that a nitrogen atom of the heterocyclic orsubstituted heterocyclic is not bound to the —C(O)-group wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

The term “aminoacyl” and the prefix “carbamoyl” or “carboxamide” or“substituted carbamoyl” or “substituted carboxamide” refer to the group—C(O)NR⁴²R⁴² where each R⁴² is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic; or where each R⁴² is joinedto form together with the nitrogen atom a heterocyclic or substitutedheterocyclic wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic are as defined herein.

The term “acyloxy” refers to the moiety —O-acyl, wherein the oxygen atomis attached to the carbonyl (—C(O)) of the acyl moiety.

The term “alkenyl” refers to a vinyl unsaturated monovalent hydrocarbylgroup having from 2 to 6 carbon atoms and preferably 2 to 4 carbonatoms, and having at least 1, and preferably from 1 to 2 sites of vinyl(>C═C<) unsaturation. Such groups are exemplified by vinyl (ethen-1-yl),allyl, but-3-enyl and the like.

The term “substituted alkenyl” refers to alkenyl groups having from 1 to3 substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic. Thisterm includes both E (cis) and Z (trans) isomers as appropriate. It alsoincludes mixtures of both E and Z components.

The term “alkynyl” refers to an acetylinic unsaturated monovalenthydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3carbon atoms and having at least 1, and preferably from 1 to 2 sites ofacetylenic (—C≡C—) unsaturation. This group is exemplified byethyn-1-yl, propyn-1-yl, propyn-2-yl, and the like.

The term “substituted alkynyl” refers to alkynyl groups having from 1 to3 substituents, and preferably 1 to 2 substituents, selected from thoselisted for substituted alkenyl.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NR⁴¹R⁴¹, where eachR⁴¹ is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclic,—SO₂-substituted heterocyclic, provided that both R⁴¹ groups are nothydrogen; or the R⁴¹ groups can be joined together with the nitrogenatom to form a heterocyclic or substituted heterocyclic ring.

The term “acylamino” refers to the group —NR⁴⁵C(O)alkyl, —NR⁴⁵C(O)substituted alkyl, —NR⁴⁵C(O)cycloalkyl, —NR⁴⁵C(O) substitutedcycloalkyl, —NR⁴⁵C(O)alkenyl, —NR⁴⁵C(O) substituted alkenyl,—NR⁴⁵C(O)alkynyl, —NR⁴⁵C(O) substituted alkynyl, —NR⁴⁵C(O)aryl,—NR⁴⁵C(O) substituted aryl, —NR⁴⁵C(O)heteroaryl, —NR⁴⁵C(O) substitutedheteroaryl, —NR⁴⁵C(O)heterocyclic, and —NR⁴⁵C(O) substitutedheterocyclic where R⁴⁵ is hydrogen or alkyl and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are defined herein.

The term “carbonyloxyamino” refers to the groups —NR⁴⁶C(O)O-alkyl,—NR⁴⁶C(O)O-substituted alkyl, —NR⁴⁶C(O)O-alkenyl, —NR⁴⁶C(O)O-substitutedalkenyl, —NR⁴⁶C(O)O-alkynyl, —NR⁴⁶C(O)O-substituted alkynyl,—NR⁴⁶C(O)O-cycloalkyl, —NR⁴⁶C(O)O-substituted cycloalkyl,—NR⁴⁶C(O)O-aryl, —NR⁴⁶C(O)O-substituted aryl, —NR⁴⁶C(O)O-heteroaryl,—NR⁴⁶C(O)O-substituted heteroaryl, —NR⁴⁶C(O)O-heterocyclic, and—NR⁴⁶C(O)O-substituted heterocyclic where R⁴⁶ is hydrogen or alkyl andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein. It should be understoodthat the group “oxycarbonylamino” refers to groups similar to those justdescribed except that the attachment of the oxygen and carbonyl(—C(═O)—) group are switched. Also, the term “oxythiocarbonylamino” aresimilar to those just described; however, a thiocarbonyl (—C(═S)—) isused in place of a carbonyl.

The term “aminocarbonyloxy” or as a prefix “carbamoyloxy” or“substituted carbamoyloxy” refers to the groups —OC(O)NR⁴⁷R⁴⁷ where eachR⁴⁷ is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic; or where each R⁴⁷ is joined to form, togetherwith the nitrogen atom a heterocyclic or substituted heterocyclic andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

The term “aminocarbonylamino” refers to the group —NR⁴⁹C(O)NR⁴⁹ whereeach R⁴⁹ is independently selected from the group consisting of hydrogenand alkyl. The term “aminothiocarbonylamino” refers to the moiety—NR⁴⁹C(S)R⁴⁹—, wherein R⁴⁹ is as defined above.

The term “aryl” or “Ar” refers to a monovalent aromatic carbocyclicgroup of from 6 to 14 carbon atoms having a single ring (e.g., phenyl)or multiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is the aryl group. Preferred aryls include phenyl andnaphthyl.

The term “substituted aryl” refers to aryl groups, as defined herein,which are substituted with from 1 to 4, preferably 1 to 3, substituentsselected from those listed for substituted alkyl with the exception thatsuch substitution does not include oxo or thioxo.

The term “aryloxy” refers to the group aryl-O— that includes, by way ofexample, phenoxy, naphthoxy, and the like.

The term “substituted aryloxy” refers to substituted aryl-O— groups.

The term “aryloxyaryl” refers to the group -aryl-O-aryl.

The term “substituted aryloxyaryl” refers to aryloxyaryl groupssubstituted with from 1 to 3 substituents on either or both aryl ringsas defined above for substituted aryl.

The term “carboxyl” refers to —COOH or salts thereof.

The term “carboxyl esters” or “alkoxycarbonyl” refers to the groups—C(O)O-alkyl, —C(O)O-substituted alkyl, —C(O)O-aryl, and—C(O)O-substituted aryl wherein alkyl, substituted alkyl, aryl andsubstituted aryl are as defined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 10carbon atoms having single or multiple cyclic rings including, by way ofexample, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl andthe like.

The term “substituted cycloalkyl” refers to a cycloalkyl group, havingfrom 1 to 5 substituents selected from those listed for substitutedalkyl.

The term “cycloalkoxy” refers to —O-cycloalkyl groups.

The term “substituted cycloalkoxy” refers to —O-substituted cycloalkylgroups.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodoand preferably is fluoro or chloro.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms, preferably from 1 to 10 carbon atoms, and 1 to 4 heteroatomsselected from the group consisting of oxygen, nitrogen and sulfur withinthe ring. Such heteroaryl groups can have a single ring (e.g., pyridinylor furyl) or multiple condensed rings (e.g., indolizinyl, benzothienyl,2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, benzofuran,2,3-dihydrobenzofuran, benzothiazole, benzooxazole, benzo[1,3]dioxolyl,and the like). Preferred heteroaryls include pyridinyl, pyrrolyl,indolyl, thiophenyl, and furyl.

The term “substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl.

The term “heteroaryloxy” refers to the group —O-heteroaryl and“substituted heteroaryloxy” refers to the group —O-substitutedheteroaryl.

The term “heterocycle” or “heterocyclic,” or “heterocyclyl” refers to asaturated or unsaturated group having a single ring or multiplecondensed rings, from 1 to 10 carbon atoms and from 1 to 4 hetero atomsselected from the group consisting of nitrogen, sulfur or oxygen withinthe ring wherein, in fused ring systems, one or more the rings can bearyl or heteroaryl provided that the point of attachment is at theheterocycle.

The term “substituted heterocyclic” or “substituted heterocyclyl” refersto heterocycle groups that are substituted with from 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydro-isoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

The term “heterocyclyloxy” refers to the group —O-heterocyclic and“substituted heterocyclyloxy” refers to the group —O-substitutedheterocyclic.

The term “thiol” refers to the group —SH.

The term “alkylthio” refer to the groups —S-alkyl where alkyl is asdefined above.

The term “substituted alkylthio” and “substituted alkylsulfanyl” referto the group —S-substituted alkyl, where substituted alkyl is as definedabove.

The term “cycloalkylthio” or “cycloalkylsulfanyl” refers to the groups—S-cycloalkyl where cycloalkyl is as defined above.

The term “substituted cycloalkylthio” refers to the group —S-substitutedcycloalkyl where substituted cycloalkyl is as defined above.

The term “arylthio” or “arylsulfanyl” refers to the group —S-aryl and“substituted arylthio” refers to the group —S-substituted aryl wherearyl and substituted aryl are as defined above.

The term “heteroarylthio” refers to the group —S-heteroaryl and“substituted heteroarylthio” refers to the group —S-substitutedheteroaryl where heteroaryl and substituted heteroaryl are as definedabove.

The term “heterocyclicthio” refers to the group —S-heterocyclic and“substituted heterocyclicthio” refers to the group —S-substitutedheterocyclic where heterocyclic and substituted heterocyclic are asdefined above.

The term “pharmaceutically acceptable salt” refers to pharmaceuticallyacceptable salts of a compound, which salts are derived from a varietyof organic and inorganic counter ions well known in the art and include,by way of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

The term “prodrug,” refers to compounds of formula I, and/or II of thisinvention that include chemical groups which, in vivo, can be convertedinto the carboxylate group on the amino-acid side chain of the compoundsand/or can be converted into the amide carbonyl group and/or can besplit off from the amide N-atom and/or can be split off from the 4-Oatom of the isoquinoline and/or can be converted into the 1-cyano groupand/or can be split off from the N-atom of the isoquinoline ring toprovide for the active drug, a pharmaceutically acceptable salt thereofor a biologically active metabolite thereof. Suitable groups are wellknown in the art and particularly include: for the carboxylic acidmoiety on the glycine or alanine substituent, a prodrug selected from,e.g., esters including, but not limited to, those derived from alkylalcohols, substituted alkyl alcohols, hydroxy substituted aryls andheteroaryls and the like; amides, particularly amides derived fromamines of the formula HNR²⁰R²¹ where R²⁰ and R²¹ are independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and thelike; hydroxymethyl, aldehyde and derivatives thereof; and for theisoquinoline N atom, a prodrug selected from, e.g., N-oxides and N-alkylderivatives.

The term “solvates,” refers to a complex formed by combination ofsolvent molecules with molecules or ions of the solute. The solvent canbe an organic compound, an inorganic compound, or a mixture of both.Some examples of solvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.

The term “tautomer” refers to alternate forms of a molecule that differin the position of a proton, such as enol-ketol and imine-enaminetautomers, or the tautomeric forms of heteroaryl groups containing aring atom attached to both a ring —NH— moiety and a ring ═N— moiety suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

C. Compositions and Methods of the Invention

The invention provides for use of a compound of formula I and/or II forthe manufacture of a medicament for use in treating various conditionsor disorders as described herein. In one embodiment, a pharmaceuticalcomposition is provided comprising a pharmaceutically acceptableexcipient or carrier, and a therapeutically effective amount of at leastone compound of formula I and/or II.

The medicament or composition can further comprise at least oneadditional therapeutic agent selected from the group including, but notlimited to, vitamin B₁₂, ferrous sulfate, folic acid, and/or recombinanterythropoietin or an erythropoiesis stimulating protein (ESP).

The compounds of the present invention, or medicaments or compositionscomprising the compounds, can be used to modulate the stability and/oractivity of HIF, and thereby activate HIF-regulated gene expression. Thecompound, or composition or medicament thereof, can be used in methodsto treat, pretreat, or delay progression or onset of conditionsassociated with HIF including, but not limited to, anemic, ischemic, andhypoxic conditions. In various embodiments, the compound is administeredimmediately following a condition producing acute ischemia, e.g.,myocardial infarction, pulmonary embolism, intestinal infarction,ischemic stroke, and renal ischemic-reperfusion injury. In anotherembodiment, the compound, or composition or medicament thereof, isadministered to a patient diagnosed with a condition associated with thedevelopment of chronic ischemia, e.g., cardiac cirrhosis, maculardegeneration, pulmonary embolism, acute respiratory failure, neonatalrespiratory distress syndrome, and congestive heart failure. In yetanother embodiment, the compound, or composition or medicament thereof,is administered immediately after a trauma or injury. In otherembodiments, the compound, or composition or medicament thereof, can beadministered to a subject based on predisposing conditions, e.g.,hypertension, diabetes, occlusive arterial disease, chronic venousinsufficiency, Raynaud's disease, chronic skin ulcers, cirrhosis,congestive heart failure, and systemic sclerosis. In still otherembodiments, compounds may be administered to pretreat a subject todecrease or prevent the development of tissue damage associated withischemia or hypoxia.

The compounds of the present invention, or compositions or medicamentsthereof, can also be used to increase endogenous erythropoietin (EPO).The compounds, or composition or medicament thereof, can be administeredto prevent, pretreat, or treat EPO-associated conditions, including,e.g., conditions associated with anemia and neurological disorders.Conditions associated with anemia include disorders such as acute orchronic kidney disease, diabetes, cancer, ulcers, infection with virus,e.g., HIV, bacteria, or parasites; inflammation, etc. Anemic conditionscan further include those associated with procedures or treatmentsincluding, e.g., radiation therapy, chemotherapy, dialysis, and surgery.Disorders associated with anemia additionally include abnormalhemoglobin and/or erythrocytes, such as found in disorders such asmicrocytic anemia, hypochromic anemia, aplastic anemia, etc.

The compounds can be used to increase endogenous EPO in a subjectundergoing a specific treatment or procedure, prophylactically orconcurrently, for example, an HIV-infected anemic patient being treatedwith azidothymidine (zidovudine) or other reverse transcriptaseinhibitors, an anemic cancer patient receiving cyclic cisplatin- ornon-cisplatin-containing chemotherapeutics, or an anemic or non-anemicpatient scheduled to undergo surgery. Additionally, the compounds can beused to increase endogenous EPO levels in an anemic or non-anemicpatient scheduled to undergo surgery to reduce the need for allogenicblood transfusions or to facilitate banking of blood prior to surgery.

The invention is also directed to use of a compound, or composition ormedicament thereof, to treat, pretreat, or delay onset of a conditionassociated with a disorder selected from the group consisting of anemicdisorders; neurological disorders and/or injuries including cases ofstroke, trauma, epilepsy, and neurodegenerative disease; cardiacischemia including, but not limited to, myocardial infarction andcongestive heart failure; liver ischemia including, but not limited to,cardiac cirrhosis; renal ischemia including, but not limited to, acutekidney failure and chronic kidney failure; peripheral vasculardisorders, ulcers, burns, and chronic wounds; pulmonary embolism; andischemic-reperfusion injury.

The invention is also directed to a method of inhibiting the activity ofat least one hydroxylase enzyme which modifies the alpha subunit ofhypoxia inducible factor. The HIF hydroxylase enzyme may be anasparaginyl hydroxylase such as Factor Inhibiting HIF (FIH); and/or aprolyl hydroxylase including, but not limited to, the group consistingof EGLN1, EGLN2, and EGLN3. The method comprises contacting the enzymewith an inhibiting effective amount of one or more compounds selectedfrom the group comprising compounds of formula I and/or II.

D. Synthesis of Compounds of the Invention

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mol ratios of reactants,solvents, pressures, etc.) are given, other process conditions can alsobe used unless otherwise stated. Optimum reaction conditions may varywith the particular reactants or solvent used, but such conditions canbe determined by one skilled in the art by routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

Furthermore, the compounds of this invention may contain one or morechiral centers. Accordingly, if desired, such compounds can be preparedor isolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included within the scope ofthis invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce orSigma (St. Louis, Mo., USA). Others may be prepared by procedures, orobvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley, and Sons, 5^(th) Edition,2001), and Larock's Comprehensive Organic Transformations (VCHPublishers Inc., 1989).

The isoquinolines 300, 1000, and 1100 of this invention can be preparedby the synthetic protocols illustrated in Scheme 1.

R¹, R², R³, R⁴, and R⁵ are as defined herein. Compound 100 (wherein PG¹refers to a suitable protecting group such as methyl, ethyl, butyl,etc.) are reacted with at least a stoichiometric amount and preferablyan excess of a suitable alpha-amino acid, compound 200 (particularly,but not limited to, glycine). The reaction is conducted underconventional coupling conditions well known in the art. In oneembodiment, the reaction is conducted in the presence of sodiummethoxide or another suitable base in methanol or another suitablesolvent under elevated reaction temperatures and particularly at reflux.The reaction is continued until it is substantially complete whichtypically occurs within about 1 to 72 h. Alternatively, the reaction canbe performed at elevated temperatures in a microwave oven. Upon reactioncompletion, the compound 300 can be recovered by conventional techniquessuch as neutralization, extraction, precipitation, chromatography,filtration and the like.

Alternatively, coupling of compound 100 (typically as the correspondingfree acid) with compound 200 (typically as ester derivatives) canproceed via conventional peptide coupling procedures well known in theart. This coupling reaction is typically conducted using well-knowncoupling reagents such as carbodiimides, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphonate) and the like. Suitable carbodiimides include, byway of example, dicyclohexylcarbodiimide (DCC),1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (DECI) and the like. Ifdesired, polymer supported forms of carbodiimide coupling reagents mayalso be used including, for example, those described in TetrahedronLetters, 34 (48), 7685 (1993). Additionally, well-known couplingpromoters, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, may be used to facilitate the coupling reaction. This couplingreaction is typically conducted by contacting the corresponding freeacids of compound 100 with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of an ester of compound 200, in an inert diluentsuch as dichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 37° C. forabout 12 to about 24 hours. Upon completion of the reaction, thecorresponding ester of compound 300 is recovered by conventional methodsincluding neutralization, extraction, precipitation, chromatography,filtration, and the like, and is then transformed into compound 300 byhydrolysis.

Alternatively, compound 100 (typically as the corresponding free acid)can be converted into an acid halide and the acid halide coupled withthe ester of compound 200 to provide for the esters of compound 300. Theacid halide of compound 100 can be prepared by contacting compound 100(typically as the corresponding free acid) with an inorganic acid halidesuch as thionyl chloride, phosphorous trichloride, phosphoroustribromide, or phosphorous pentachloride, or, particularly, with oxalylchloride under conventional conditions. Generally, this reaction isconducted using about 1 to 5 molar equivalents of the inorganic acidhalide or oxalyl chloride, either neat or in an inert solvent such asdichloromethane or carbon tetrachloride, at temperature in the range ofabout 0° C. to about 80° C. for about 1 to about 48 hours. A catalyst,such as DMF, may also be used in this reaction.

The acid halide (not shown) is then contacted with at least oneequivalent, preferably about 1.1 to about 1.5 equivalents, of an esterof compound 200, in an inert diluent such as dichloromethane, at atemperature ranging from about −70° C. to about 40° C. for about 1 toabout 24 hours. Preferably, this reaction is conducted in the presenceof a suitable base to scavenge the acid generated during the reaction.Suitable bases include, by way of example, tertiary amines such astriethylamine, diisopropylethylamine, N-methyl-morpholine, and the like.Alternatively, the reaction can be conducted under Schotten-Baumann-typeconditions using aqueous alkali such as sodium hydroxide and the like.Upon completion of the reaction, the ester of compound 300 is recoveredby conventional methods including neutralization, extraction,precipitation, chromatography, filtration, and the like, and is thentransformed into compound 300 by hydrolysis.

Compound 100 may be obtained by cyanation of compound 500 (wherein X isCl, Br, or I; and PG¹ refers to a suitable protecting group such asmethyl, ethyl, butyl, etc.) with a suitable cyanide source such as, byway of example, CuCN, Zn(CN)₂, etc. The cyanation may occur in thepresence of a suitable catalyst such as, by way of example, palladiumcatalyst such as tris(dibenzylideneacetone)dipalladium(0) (see, e.g.,Sundermeier et al. (2003) Eur. J. Inorg. Chem. 2003 (19):3513-3526)and/or additives using a suitable solvent such as, by way of example,DMF or N,N-dimethylacetamide. Upon reaction completion, compound 100 canbe recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation. Compound 100 obtained by this method may be further alteredby modifying its R¹, R², R³, and/or R⁴ moieties, e.g., if R³ is OCH₂Ph,the R³ moiety can be transformed into OH using conventional reductiontechniques (particularly by hydrogenation catalyzed by palladium oncarbon, etc.).

Alternatively, compound 400 (wherein X is selected from Cl, Br, or I)may be reacted with a suitable cyanide source such as, by way ofexample, CuCN, Zn(CN)₂, etc. to give compound 300. The cyanation may becarried out as described above. Upon reaction completion, compound 300can be recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation. Compound 400 may be obtained by reaction between compound 500(wherein X is selected from Cl, Br, or I; and PG¹ refers to a suitableprotecting group such as methyl, ethyl, butyl, etc.) and compound 200 inanalogy to the corresponding reactions between compound 100 and compound200 (Scheme 1; also, see U.S. Pat. No. 6,093,730 and U.S. PatentApplication Publication No. 2006/217416, which is hereby incorporated byreference).

Alternatively, the 4-hydroxy group of compound 500 can be alkylated withsuitable reagents such as, by way of example, alkyl halides, alkylsulfates, benzyl halides, diazo compounds, etc. The alkylation may takeplace in the presence of suitable base such as Cs₂CO₃ and/or additivesusing a suitable solvent such as, by way of example, DMF to givecompound 600 (wherein PG² is preferably but not limited to, methyl).Upon reaction completion, compound 600 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like; or, alternatively, used in thenext step without purification and/or isolation.

Compound 600 (wherein X is selected from Cl, Br, or I) can be reactedwith a suitable cyanide source such as, by way of example, CuCN,Zn(CN)₂, etc., to give compound 700. The cyanation may be carried out asdescribed above. Hydrolysis of compound 700 using conventional standardconditions provides compound 800. Upon reaction completion, compound 800can be recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation.

Coupling of compound 800 with compound 900 (wherein PG³ refers to asuitable protecting group such as methyl, ethyl, butyl, etc.; preformedor generated in situ from its salts by addition of a suitable base) canproceed via conventional peptide coupling procedures well known in theart and as described above. The coupling reaction is typically conductedby contacting compound 800 with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of compound 900, in an inert diluent such asdichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like and, if required, in the presence ofa suitable base. Generally, this reaction is conducted at a temperatureranging from about 0° C. to about 37° C. for about 12 to about 24 hours.Upon completion of the reaction, compound 1000 is recovered byconventional methods including neutralization, extraction,precipitation, chromatography, filtration, and the like, and can then betransformed into compound 1100 by hydrolysis.

Alternatively, compound 800 can be converted into an acid halide and theacid halide coupled with compound 900 to provide compound 1000. The acidhalide of compound 800 can be prepared by contacting compounds 800 withan inorganic acid halide or with oxalyl chloride under conventionalconditions, e.g., as described above. The acid halide (not shown) isthen contacted with at least one equivalent, preferably about 1.1 toabout 1.5 equivalents of compound 900 in an inert diluent such asdichloromethane, at a temperature ranging from about −70° C. to about40° C. for about 1 to about 24 hours. Preferably, this reaction isconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include tertiary aminessuch as triethylamine, diisopropylethylamine, N-methyl-morpholine, andthe like. Alternatively, the reaction can be conducted underSchotten-Baumann-type conditions using aqueous alkali such as sodiumhydroxide and the like. Upon completion of the reaction, compound 1000is recovered by conventional methods including neutralization,extraction, precipitation, chromatography, filtration, and the like, andcan then be transformed into compound 1100 by hydrolysis (Scheme 1).

Compound 500, for use in the reactions above, can be prepared accordingto synthetic routes as depicted in Scheme 2.

R¹, R², R³, and R⁴ are as defined herein. Treatment of compound 1200with phosphorous oxychloride or phosphorous oxybromide using a suitablesolvent such as acetonitrile or toluene particularly at refluxtemperature gives compound 500 wherein X is Cl or Br, respectively. Thereaction typically occurs within about 1 to 72 h. Alternatively, thereaction can be performed at elevated temperatures in a microwave oven.Upon reaction completion, compound 500 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like; or, alternatively, used in thenext step without purification and/or isolation.

Alternatively, compound 1300 can be halogenated using conventionalmethods to give compound 500 wherein X is Cl, Br, or I. The halogenationof compound 1300 can be performed with a stoichiometric amount or slightexcess of, e.g., N-bromosuccinimide in the presence of a catalyticamount of benzoylperoxide, azobisisobutyronitrile, or another suitablefree radical initiator, in CCl₄, benzene, or another suitable solventknown to one skilled in the art typically at reflux temperature orhigher temperatures using a microwave oven. Upon reaction completion,compounds 500 can be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like; or, alternatively, used in the next step withoutpurification and/or isolation. Alternatively, compound 1300 can beobtained by halogenating compound 1200 as described above followed byreduction using conventional methods such as hydrogenation catalyzed bypalladium on carbon, etc.

The synthesis of substituted isoquinoline carboxylic acids are generallyknown in the art and are described by, for example, Weidmann, et al.,U.S. Pat. No. 6,093,730, which is incorporated herein by reference inits entirety. Compound 1200, for use in the reactions above, can beobtained using the methods outlined in Scheme 3 (see US2006/217416,which is hereby incorporated by reference).

R¹, R², R³, and R⁴ are as defined herein. Compound 1200 can be obtainedfrom compound 1500 (particularly wherein R is methyl, ethyl, or butyl)by Gabriel-Colman rearrangement with a solution of 2 equivalents ofsodium in an appropriate alcohol such as n-butanol at an elevatedtemperature of about 95-100° C. for about 1 to 4 h. Compound 1500 iseither commercially available or are easily obtained by reacting thephthalic acid, compound 1400 or its derivatives (particularly thecorresponding acid anhydrides) with an equimolar amount of glycine orsalts of glycine esters (particularly hydrochloride salts of glycinemethyl, ethyl or butyl ester) neat typically at 150 to 250° C. for 15 to90 min until no water is released. If glycine is used, the product (R═H)is esterified using conventional methods, e.g., by refluxing in asuitable alcohol such as methanol or ethanol in the presence of aconcentrated sulfuric acid, etc. Compound 1400 and its derivatives,e.g., the corresponding acid anhydrides, are either commerciallyavailable or are easily accessible by reacting compound 1600 with anexcess of phenol ArOH in a suitable solvent such as, e.g., DMF, in thepresence of a base such as, by example, an excess of potassiumcarbonate. The resulting compound 1700 is readily hydrolyzed byrefluxing in a solution of KOH in water/methanol (typically 1 to 3 days)to give the compound 1400.

Alternatively, compound 1400 can be obtained by analogous methods fromcompound 1800. Reaction between compound 1800 and phenol ArOH providescompound 1900, which can be readily hydrolyzed to the correspondingphthalic acid, compound 1400. Biphenyl-2,3-dicarboxylic acid can beobtained by oxidation of compound 2000 with potassium permanganate inthe presence of a phase transfer catalyst. Alternatively, compound 1500can be obtained by reacting compound 2100 with an excess of a phenolatesuch as, by way of example, sodium phenolates ArONa, typically byheating in DMF. The resulting phthalimide, compound 2200, can betransformed to compound 1500 by N-alkylation with an excess of ahaloacetic acid ester such as, by way of example, the methyl or ethylesters of bromoacetic acid, using conventional methods such as refluxingin a suitable solvent such as acetone in the presence of a suitable basesuch as potassium carbonate. Compound 1500 can be obtained analogouslyby N-alkylation of commercially available phthalimides, compound 2400.Alternatively, compound 1500 can be obtained by N-alkylation of compound2100 using methods as described above. The resulting compound 2300 canbe transformed to compound 1500 by reaction with phenols ArOH in asuitable solvent, such as dimethylacetamide at 105° C., in the presenceof a suitable base such as potassium carbonate. Alternatively, compound1500 with a phenolic OH group can be transformed to the correspondingphenol ethers by O-alkylation using conventional methods such as, by wayof example, refluxing with electrophiles such as methyl iodide (MeI),isopropyl iodide (iPrI), butyl iodide (BuI), etc. in the presence of abase or by alkylating the phenolic OH group with an alcohol such ascyclohexanol using a variant of the Mitsunobu reaction.

Compound 1300, for use in the reactions above, can be obtained using themethods outlined in Scheme 4 (see also US2006/217416).

R¹, R², R³, and R⁴ are as defined herein. Compound 2500 (particularlywherein R is methyl or ethyl, and X is Cl, Br, or I; preformed orgenerated in situ by anion exchange using sodium iodide) is reacted witha protected glycine ester, compound 2600 (particularly wherein PG¹ ismethyl or ethyl and PG⁴ is toluene-4-sulfonyl or 2,4-dimethoxybenzyl) inthe presence of a suitable base such as potassium carbonate, optionallyin the presence of sodium iodide, in a suitable solvent such as DMF at,e.g., room temperature for about 2 to 24 h. Upon reaction completion,the resulting compound 2700 can be recovered by conventional techniquessuch as neutralization, extraction, precipitation, chromatography,filtration and the like; or, alternatively, used in the next stepwithout purification and/or isolation. Alternatively, compound 2700 witha phenolic OH group can be transformed to the corresponding phenolethers by O-alkylation using conventional methods such as, by way ofexample, refluxing with electrophiles such as benzyl bromide, etc. inthe presence of a base.

Compound 2700 wherein PG⁴ is, e.g., 2,4-dimethoxybenzyl, can be cyclizedto compound 2800 by treatment with a suitable base such as, by way ofexample, 2 equivalents of potassium tert-butoxide, in THF at 0° C. forabout 1 h and then room temperature for about 3 h. Upon reactioncompletion, the resulting compound 2800 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like; or, alternatively, used in thenext step without purification and/or isolation. Compound 2800 is thentreated with thionyl chloride, e.g., about 1.5 equivalents, in asuitable solvent such as dichloromethane at 0° C. for about 1 h and thenroom temperature for about 3 h. Upon reaction completion, the resultingcompound 1300 can be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like; or, alternatively, used in the next step withoutpurification and/or isolation. Alternatively, compound 2700 wherein PG⁴is, e.g., toluene-4-sulfonyl, can be cyclized by treatment with asuitable base such as, by way of example, 3 to 4 equivalents of sodiummethoxide in methanol at room temperature for 3 h to 3 d. Upon reactioncompletion, the resulting compound 1300 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like; or, alternatively, used in thenext step without purification and/or isolation.

Compound 1300 can be further altered by modifying its R¹, R², R³, and/orR⁴ moieties. For example, R² is SPh then the R² moiety can betransformed into SO₂Ph using conventional oxidation techniques such as,by way of example, treatment with m-chloroperoxybenzoic acid inmethylenechloride at room temperature. Alternatively, if R⁴ is, e.g.,iodine, the iodine atom can be substituted with phenols ArOH such as,e.g., ortho-, metha-, and para-methoxyphenol, to give the corresponding8-aryloxy isoquinolines by reacting the iodo-isoquinoline with ArOH,e.g., 5 equivalents in a suitable solvent such as DMF at refluxtemperature in the presence of a suitable base, such as 5 equivalent ofcesium carbonate, and a suitable catalyst, such as 1 equivalent of CuCland 0.4 equivalents of 2,2,6,6-tetramethyl-heptane-3,5-dione, for about15 min. Upon reaction completion, the resulting compound 1300 can berecovered by conventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation.

Compound 2500, for use in the reactions described above, can be obtainedusing the methods outlined in Scheme 5 (see also US2006/217416).

R¹, R², R³, and R⁴ are as defined herein. Compound 2500, particularlyortho-toluic acid esters such as 3100, 3300, 3400, or 3800 wherein R is,e.g., methyl or ethyl, can be halogenated, by way of example, byaddition of a stoichiometric amount or a slight excess ofN-bromosuccinimide in the presence of a catalytic amount ofbenzoylperoxide, azobisisobutyronitrile, or another suitable freeradical initiator in CCl₄, benzene or another suitable solvent at, e.g.,reflux temperature. Upon reaction completion, compound 2500 can berecovered by conventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation.

Compound 3100 is accessible typically by reacting a mixture of thesodium salt of compound 2900, a slight excess of a sodium phenolateArONa, a catalytic amount of copper bronze, and 1,2-dichlorobenzene at,e.g., reflux temperature for about 2 h. Upon reaction completion,compound 3000 can be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like; or, alternatively, used in the next step withoutpurification and/or isolation. Compounds 3000 and 3200 are easilyconverted to the corresponding esters, compounds 3100 and 3300,respectively, using conventional methods such as refluxing in a suitablealcohol such as methanol or ethanol in the presence of concentratedsulfuric acid; or refluxing together with an alkylating reagent such asdimethylsulfate in the presence of a base such as potassium carbonate ina suitable solvent such as diethylketone. Compound 3400 can be obtainedby adding compound 3300, an excess of phenol, an excess of cesiumcarbonate, and an excess of 1-naphthoic acid, molecular sieves, acatalytic amount of ethyl acetate, and an appropriate solvent such asanhydrous toluene to a catalytic amount of copper(I)trifluoromethanesulfonate-benzene complex at room temperature. Themixture is refluxed under nitrogen for about 2 days before it is cooledto room temperature and filtered. Compound 3400 can be recovered byconventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation. Alternatively, a mixture of compound 3300, an excess of anappropriately substituted phenol, an excess of cesium carbonate, anexcess of 2,2,6,6-tetramethylheptane-3,5-dione, and copper (I) chloridein N-methyl-2-pyrrolidone can be heated at 100 to 150° C. for 1-5 days,cooled to room temperature, and quenched with water. Compound 3400 canthen be recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation. Compound 3800 are easily accessible by treatment of 3700 withtert-butyl-dimethylsilylchloride.

Alternatively, compound 2500 is obtained by reaction of appropriatelysubstituted phthalides such as compound 3600 with suitable halogenatingreagents such as, by way of example, 1.3 equivalents of thionylchloridein the presence of suitable catalysts such as boric acid andtriphenylphosphine oxide at 130 to 140° C. for about 18 h followed bytreatment with an appropriate alcohol such as methanol. Compound 2500can be recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like; or,alternatively, used in the next step without purification and/orisolation. Compound 3600 is easily obtained by reacting 3500 withbenzylzinc bromide using a variant of the Negishi coupling.

E. Compound Testing

The biological activity of the compounds of the invention may beassessed using any conventionally known methods. Suitable assay methodsare well known in the art. The following assays are presented only asexamples and are not intended to be limiting. The compounds of theinvention are active in at least one of the following assays.

It is contemplated that due to the cyano substitution at the C-1position of the isoquinoline compounds, the compounds of the inventionshow surprising and unexpected improved efficacy over comparablecompounds not possessing cyano at the C-1 position. For example,compounds having a cyano at the C-1 position of the isoquininoline ring,when compared to corresponding compounds having, for example, hydrogen,methyl, or halogen, at the C-1 position are more than 2-fold, moreparticularly, more than 4-fold, even more particularly more than 10-foldor 50-fold more potent at increasing circulating erythropoietin levels.Examples of this are more thoroughly discussed in Example 54.

a. Cell-Based HIFα Stabilization Assay

Human cells derived from various tissues are separately seeded into 35mm culture dishes and grown at 37° C., 20% O₂, 5% CO₂ in standardculture medium, e.g., DMEM, 10% FBS. When cell layers reach confluence,the media is replaced with OPTI-MEM media (Invitrogen Life Technologies,Carlsbad Calif.) and cell layers are incubated for approximately 24hours in 20% O₂, 5% CO₂ at 37° C. Compound or 0.013% DMSO is then addedto existing medium, and incubation is continued overnight.

Following incubation, the media is removed, centrifuged, and stored foranalysis (see VEGF and EPO assays below). The cells are washed two timesin cold phosphate buffered saline (PBS) and then lysed in 1 mL of 10 mMTris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL (Sigma-Aldrich, St.Louis Mo.), and a protease inhibitor mix (Roche Molecular Biochemicals)for 15 minutes on ice. Cell lysates are centrifuged at 3,000×g for 5minutes at 4° C., and the cytosolic fractions (supernatant) arecollected. The nuclei (pellet) are resuspended and lysed in 100 μL of 20mM HEPES (pH 7.2), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and aprotease mix (Roche Molecular Biochemicals), centrifuged at 13,000×g for5 minutes at 4° C., and the nuclear protein fractions (supernatant) arecollected.

Nuclear fractions are analyzed for HIF-1α using a QUANTIKINE immunoassay(R&D Systems, Inc., Minneapolis Minn.) according to the manufacturer'sinstructions.

b. Cell-Based VEGF and EPO ELISA Assays

Conditioned media collected from cell cultures as described above isanalyzed for vascular endothelial growth factor (VEGF) and/orerythropoietin (EPO) expression using an appropriate QUANTIKINEimmunoassay (R&D Systems) according to the manufacturer's instructions.

c. HIF-PH Assay

Ketoglutaric acid α-[1-¹⁴C]-sodium salt, Ketoglutaricacid-[1-¹⁴C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLCpurified peptide may be obtained from commercial sources, e.g.,Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (DublinCalif.), respectively. Peptides for use in the assay may be fragments ofHIFα as described above or as disclosed in International Publication WO2005/118836, incorporated by reference herein. HIF-PH, e.g., HIF-PH2(EGLN1) can be expressed in, e.g., insect Hi5 cells and partiallypurified, e.g., through a SP ion exchange chromatography column. Enzymeactivity is determined by capturing ¹⁴CO₂ using an assay described byKivirikko and Myllyla (1982, Methods Enzymol 82:245-304). Assayreactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acidsodium salt, 0.30 μCi/mL ketoglutaric acid α-[1-¹⁴C]-sodium salt; 40 μMFeSO₄, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μMpeptide substrate and various concentrations of compound of theinvention. Reactions are initiated by addition of HIF-PH enzyme.

The peptide-dependent percent turnover is calculated by subtractingpercent turnover in the absence of peptide from percent turnover in thepresence of substrate peptide. Percent inhibition and IC₅₀ arecalculated using peptide-dependent percent turnover at given inhibitorconcentrations. Calculation of IC₅₀ values for each inhibitor isconducted, e.g., using GraFit software (Erithacus Software Ltd., SurreyUK).

The compounds of the present invention when tested in assays,demonstrated improved activity as compared to non-cyano compounds, asshown in Table 1 below. Table 1 can be found in example 54.

F. Pharmaceutical Formulations and Routes of Administration

The compounds of the present invention can be delivered directly or inpharmaceutical compositions along with suitable carriers or excipients,as is well known in the art. Various treatments embodied herein cancomprise administration of an effective amount of a compound of theinvention to a subject in need, e.g., a subject having or at risk foranemia due to, e.g., chronic renal failure, diabetes, cancer, AIDS,radiation therapy, chemotherapy, kidney dialysis, or surgery. In apreferred embodiment, the subject is a mammalian subject, and in a mostpreferred embodiment, the subject is a human subject.

An effective amount of a compound can readily be determined by routineexperimentation, as can the most effective and convenient route ofadministration and the most appropriate formulation. Variousformulations and drug delivery systems are available in the art. See,e.g., Gennaro, A. R., ed. (1995) Remington's Pharmaceutical Sciences,supra.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, nasal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Thecompound or composition thereof may be administered in a local ratherthan a systemic manner. For example, a compound or composition thereofcan be delivered via injection or in a targeted drug delivery system,such as a depot or sustained release formulation.

The pharmaceutical compositions of the present invention may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Asnoted above, the compositions of the present invention can include oneor more physiologically acceptable carriers such as excipients andauxiliaries that facilitate processing of active molecules intopreparations for pharmaceutical use.

Proper formulation is dependent upon the route of administration chosen.For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In a preferred embodiment of the presentinvention, the present compounds are prepared in a formulation intendedfor oral administration. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject. The compoundsmay also be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Also, wetting agents such as sodium dodecyl sulfate may beincluded.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In one embodiment, the compounds of the present invention can beadministered transdermally, such as through a skin patch, or topically.In one aspect, the transdermal or topical formulations of the presentinvention can additionally comprise one or multiple penetrationenhancers or other effectors, including agents that enhance migration ofthe delivered compound. Transdermal or topical administration could bepreferred, for example, in situations in which location specificdelivery is desired.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, for example,gelatin, for use in an inhaler or insufflator may be formulated. Thesetypically contain a powder mix of the compound and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions or other compositions in water-soluble form.

Suspensions of the active compounds may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions of the present invention may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thepresent compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Suitable carriers for the hydrophobic molecules of the invention arewell known in the art and include co-solvent systems comprising, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. The co-solvent system may be the VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in watersolution. This co-solvent system is effective in dissolving hydrophobiccompounds and produces low toxicity upon systemic administration.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied. For example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80, the fraction size of polyethyleneglycol may be varied, other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone, and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Liposomal delivery systemsare discussed above in the context of gene-delivery systems. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using sustained-release systems, such assemi-permeable matrices of solid hydrophobic polymers containing theeffective amount of the composition to be administered. Varioussustained-release materials are established and available to those ofskill in the art. Sustained-release capsules may, depending on theirchemical nature, release the compounds for a few weeks up to over 100days. Depending on the chemical nature and the biological stability ofthe therapeutic reagent, additional strategies for protein stabilizationmay be employed.

For any composition used in the various treatments embodied herein, atherapeutically effective dose can be estimated initially using avariety of techniques well known in the art. For example, in a cellculture assay, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from cell culture assaysand other animal studies.

A therapeutically effective dose of a compound refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, which can beexpressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit hightherapeutic indices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methods knownin the art, in view of the specifics of a subject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to modulate adesired parameter, e.g., endogenous erythropoietin plasma levels, i.e.minimal effective concentration (MEC). The MEC will vary for eachcompound but can be estimated from, for example, in vitro data. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. Compounds or compositions thereof should beadministered using a regimen which maintains plasma levels above the MECfor about 10-90% of the duration of treatment, preferably about 30-90%of the duration of treatment, and most preferably between 50-90%. Incases of local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.Alternatively, modulation of a desired parameter, e.g., stimulation ofendogenous erythropoietin, may be achieved by 1) administering a loadingdose followed by a maintenance dose, 2) administering an induction doseto rapidly achieve the desired parameter, e.g., erythropoietin levels,within a target range, followed by a lower maintenance dose to maintain,e.g., hematocrit, within a desired target range, or 3) repeatedintermittent dosing.

The amount of compound or composition administered will, of course, bedependent on a variety of factors, including the sex, age, and weight ofthe subject being treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of conditions, disorders, or diseases in which anemia is amajor indication.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein,and are specifically contemplated.

EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

-   -   bs=broad singlet    -   DMSO=dimethyl sulfoxide    -   d=doublet    -   dd=doublet of doublets    -   dppf 1,1′-bis(diphenylphosphino)ferroceno    -   DMF=dimethyl formamide    -   DMEM=Dulbecco's Modified Eagle Medium    -   EtOAc=ethyl acetate    -   EDTA=ethylenediaminetetraacetic acid    -   FBS=fetal bovine serum    -   g=gram    -   h=hour    -   HPLC=High Performance Liquid Chromatography    -   Hz=hertz    -   MS=Mass Spectroscopy    -   MeONa=sodium methoxide    -   MeOH=methanol    -   MHz=mega Hertz    -   μM=micromolar    -   μL=microliter    -   mg=milligram    -   mL=milliliter    -   mM=millimolar    -   mm=millimeter    -   mmol=millimolar    -   min=minute    -   M=molar    -   mol=moles    -   m=multiplet    -   N=normal    -   NMR=nuclear magnetic resonance    -   Pd/C=palladium over carbon    -   Pd₂(dba)₃=tris(dibenzylideneacetone)dipalladium(0)    -   q=quartet    -   s=singlet    -   Ts=toluene-4-sulfonyl    -   t=triplet

Example 1 [(1-Cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butylester (1.621 g, 5 mmol; prepared as shown in Scheme 2, according to US2004/0254215 A1; ¹H NMR (200 MHz, CD₃OD) δ 11.89 (s, 1H), 8.41 (m, 1H),8.25 (m, 1H), 7.84 (m, 2H), 4.49 (t, J=7.0 Hz, 2H), 1.87 (m, 2H), 1.47(m, 2H), 1.00 (t, J=7.2 Hz, 3H)), CuCN (905 mg, 10 mmol) anddimethylformamide (20 mL) was refluxed with stirring under nitrogen for5 min. After cooling to ambient temperature the mixture was diluted withwater (300 mL). Then ethyl acetate (150 mL) was added and the mixturewas shaken thoroughly for 5 min before it was filtered through a pad ofcelite. The organic phase of the filtrate was separated, and dried overMgSO₄ before silica gel was added. The mixture was concentrated invacuo. The residue was added on top of a short column filled with silicagel. Elution with dichloromethane gave the title compound as a yellowishsolid (627 mg); MS-(−)-ion: M−1=269.2.

b. [(1-Cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

A mixture of 1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butylester (89 mg, 0.33 mmol), glycine (375 mg, 5 mmol), and a 0.5 N solutionof MeONa in MeOH (8 mL, 4 mmol) was refluxed with stirring for 48 hbefore it was concentrated in vacuo. The residue was dissolved in water(20 mL). The solution was washed with diethyl ether before its pH wasadjusted to 2 to 3 by addition of aqueous 6 N HCl. The resultingsuspension was extracted with ethyl acetate (1×30 mL). The organic phasewas dried over MgSO₄ and concentrated in vacuo to give the titlecompound as a yellowish solid (72 mg); MS-(−)-ion: M−1=270.2.

Example 22-(S)-[(1-Cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionic acid

A mixture of 1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butylester (135 mg, 0.5 mmol, see Example 1(a)), (S)-alanine (225 mg, 2.5mmol) and a 0.5 N solution of MeONa in methanol (5 mL, 2.5 mmol) washeated in a microwave oven with stirring for 40 min at 120° C. beforethe mixture was concentrated in vacuo. To the residue was added water(10 mL) and the mixture was washed with diethyl ether (4×40 mL). The pHof the purified solution was adjusted to about 2 by the addition ofaqueous 6 N HCl. The resulting suspension was extracted with ethylacetate (1×40 mL). The organic phase was dried over MgSO₄ andconcentrated in vacuo to give the title compound as a yellowish solid(101 mg); MS-(−)-ion: M−1=284.1.

Example 3[(1-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butylester

A mixture of 1-bromo-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acidbutyl ester (624 mg, 1.5 mmol; prepared as shown in Scheme 2, andaccording to US 2004/0254215 A1, ¹H NMR (CDCl₃): δ=11.89 (s, 1H), 8.35(d, 1H), 7.63 (d, 1H), 7.08 to 7.52 (m, 6H), 4.47 (t, 2H), 1.84 (m, 2H),1.48 (m, 2H), 0.99 (t, 3H)), CuCN (271 mg, 3 mmol) and dimethylformamide(6 mL) was refluxed with stirring under nitrogen for 15 min. Aftercooling to ambient temperature the mixture was diluted with ethylacetate (100 mL). The resulting suspension was filtered through a pad ofcelite. The filtrate was washed with water (2×250 mL), and dried overMgSO₄ before silica gel was added. Subsequently, the mixture wasconcentrated in vacuo. The residue was added on top of a short columnfilled with silica gel. Elution with dichloromethane gave the titlecompound as a white solid (313 mg); MS-(−)-ion: M−1=361.2.

b. [(1-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained from1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl esterand glycine analogous to Example 1(b)); MS-(−)-ion: M−1=362.0.

Example 42-(S)-[(1-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

The title compound was obtained from1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(see Example 3a) and (S)-alanine in analogy to Example 2; MS-(−)-ion:M−1=376.0.

Example 52-(R)-[(1-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

The title compound was obtained from1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid butyl ester(see Example 3a) and (R)-alanine in analogy to Example 2; MS-(−)-ion:M−1=376.1.

Example 6{[1-Cyano-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 1-Cyano-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

This compound was synthesized from1-bromo-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (prepared as shown in Scheme 2, according to US 2004/0254215A1, ¹H NMR (CDCl₃) δ=11.89 (s, 1H), 8.36 (d, 1H), 7.57 (d, 1H), 7.44 to7.50 (m, 1H), 7.08 to 7.16 (m, 4H), 4.47 (t, 2H), 1.78 to 1.93 (m, 2H),1.38 to 1.58 (m, 2H), 0.99 (t, 3H)) and CuCN in analogy to Example3(a)); MS-(−)-ion: M−1=379.2.

b.{[1-Cyano-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was obtained from1-cyano-7-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester and glycine in analogy to example 1b); MS-(−)-ion:M−1=380.0.

Example 7[(1-Cyano-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylicacid butyl ester

This compound was synthesized from1-bromo-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester (prepared as shown in Scheme 2, according to US 2004/0254215 A1,¹H NMR (CDCl₃): δ=11.96 (s, 1H), 8.52 to 8.56 (m, 2H), 7.99 (dd, 1H),4.51 (t, 2H), 1.86 (m, 2H), 1.48 (m, 2H), 1.00 (t, 3H)) and CuCN inanalogy to Example 1(a)); MS-(−)-ion: M−1=337.1.

b.[(1-Cyano-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained from1-cyano-4-hydroxy-7-trifluoromethyl-isoquinoline-3-carboxylic acid butylester and glycine in analogy to Example 1(b)); MS-(−)-ion: M−1=338.0.

Example 8[(7-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida. 7-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

This compound was synthesized from1-bromo-7-chloro-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester(prepared as shown in Scheme 2, according to US 2004/0254215 A1, ¹H NMR(CDCl₃): δ=11.92 (s, 1H), 8.34 (d, 1H), 8.25 (d, 1H), 7.75 (dd, 1H),4.49 (t, 2H), 1.86 (m, 2H), 1.48 (m, 2H), 1.00 (t, 3H)) and CuCN inanalogy to Example 1(a)); MS-(−)-ion: M−1=303.2.

b. [(7-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained from7-chloro-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butyl esterand glycine in analogy to Example 1(b)); MS-(−)-ion: M−1=303.9

Example 9[(1-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 3-Iodo-2-methyl-benzoic acid ethyl ester

To a stirred solution of 3-iodo-2-methyl-benzoic acid (30 g, 0.11 mol)in ethanol (425 mL) was added thionyl chloride (42 mL, 0.57 mol) at 0°C. The mixture was refluxed for 4.5 h before it was cooled to roomtemperature and concentrated in vacuo. The residue was partitionedbetween ethyl ether and saturated sodium bicarbonate solution. Theorganic layer was washed with brine, dried over anhydrous sodium sulfateand was concentrated in vacuo to give the title compound as a paleyellow oil (32.28 g): ¹H NMR (CDCl₃, 200 MHz): δ=7.94 (d, J=7.8 Hz, 1H),7.70 (d, J=7.4 Hz, 1H), 6.90 (t, J=7.8 Hz, 1H), 4.35 (q, J=7.2 Hz, 2H),2.65 (s, 3H), 1.38 (t, J=7.0 Hz, 3H).

b. 2-Methyl-3-phenoxy-benzoic acid ethyl ester

To a mixture of 3-iodo-2-methyl-benzoic acid ethyl ester (30.04 g, 0.10mol), phenol (14.62 g, 0.16 mol), cesium carbonate (50.6 g, 0.16 mol),1-naphthoic acid (26.7 g, 0.16 mol), molecular sieves (25.6 g, 4angstrom, 4-8 mesh), ethyl acetate (505 μA, 5 mmol), and anhydroustoluene (108 mL) was added a copper(I) trifluoromethanesulfonate-benzenecomplex (5.21 g, 0.01 mol) at room temperature. The mixture was refluxedunder nitrogen for 43 h before it was cooled to room temperature andfiltered. The filter cake was suspended in ethyl acetate (250 mL) andthe slurry was stirred for 0.5 h. The solid components were thenseparated by filtration and discarded. The filtrates were combined,washed with water, aqueous 0.5 N sodium hydroxide solution (2×), andbrine, dried over anhydrous sodium sulfate and concentrated in vacuo.The residue was purified by flash column chromatography on silica gelwith a gradient of ethyl acetate and hexanes. Fractions containing boththe title compound and the starting material 3-iodo-2-methyl-benzoicacid ethyl ester were pooled and concentrated in vacuo to give a yellowoil (15.9 g); fractions containing only the title compound were pooledand concentrated in vacuo to give a yellow oil (5.25 g): ¹H NMR (CDCl₃,200 MHz): δ=7.63 (m, 1H), 7.24 (m, 3H), 7.05 (m, 2H), 6.87 (m, 2H), 4.37(q, J=7.0 Hz, 2H), 2.45 (s, 3H), 1.40 (t, J=7.0 Hz, 3H).

c. 2-Bromomethyl-3-phenoxy-benzoic acid ethyl ester

A mixture of 2-methyl-3-phenoxy-benzoic acid ethyl ester (5.23 g, 0.02mol), N-bromosuccinimide (3.82 g, 0.02 mol) and benzoyl peroxide (247.5mg, 1.1 mmol) in carbon tetrachloride (80 mL) was refluxed for 4 hbefore it was cooled to room temperature and filtered. The filtrate waswashed with water, saturated aqueous sodium bicarbonate solution, andbrine before it was dried over anhydrous sodium sulfate and concentratedin vacuo to give the title compound as a yellow oil (7.08 g): ¹H NMR(CDCl₃, 200 MHz): δ=7.66 (m, 1H), 7.24 (m, 3H), 7.03 (m, 1H), 6.98 (m,3H), 5.09 (s, 3H), 4.42 (q, J=7.2 Hz, 2H), 1.44 (t, J=7.2 Hz, 3H).

d.2-{[Methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-3-phenoxy-benzoicacid ethyl ester

A mixture of 2-bromomethyl-3-phenoxy-benzoic acid ethyl ester (6.83 g,0.02 mol), (toluene-4-sulfonylamino)-acetic acid methyl ester (4.97 g,0.02 mol), sodium iodide (4.59 g, 0.03 mol), potassium carbonate (4.24g, 0.03 mol), and anhydrous dimethylformamide (50 mL) was stirred atroom temperature for 4 h before it was diluted with water and extractedwith ethyl acetate. The organic layer was separated and washed withwater and brine. Then it was dried over anhydrous sodium sulfate andconcentrated in vacuo. The residue was purified by flash columnchromatography on silica gel with a gradient of ethyl acetate andhexanes to give the title compound as a yellow oil (5.10 g): MS: (+) m/z497.8 (M+1).

e. 4-Hydroxy-8-phenoxy-isoquinoline-3-carboxylic acid methyl ester

To a stirred solution of2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-3-phenoxy-benzoicacid ethyl ester (5.07 g, 0.01 mol) in anhydrous methanol (22 mL) wasadded dropwise a mixture of sodium methoxide solution (30% wt, 5.6 mL)and anhydrous methanol (4 mL) at 0° C. The mixture was stirred at 0° C.for 10 minutes and then at room temperature for additional 3 h before itwas concentrated in vacuo. Water was added and the pH of the slurry wasadjusted with aqueous 1 N HCl to pH=10. The precipitate formed wasseparated by filtration, washed with water, saturated sodium bicarbonatesolution, and water before it was dried in vacuo to give the titlecompound as a white solid (2.21 g): ¹H NMR (CDCl₃, 200 MHz): δ=11.73 (s,1H), 9.21 (s, 1H), 8.06 (d, J=8.2 Hz, 1H), 7.61 (t, J=8.0 Hz, 1H), 7.40(m, 2H), 7.13 (m, 4H), 4.10 (s, 3H).

f. 1-Bromo-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acid methylester

A mixture of 4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acid methylester (103 mg, 0.35 mmol), N-bromosuccinimide (65 mg, 0.37 mmol),benzoyl peroxide (4.2 mg, 0.02 mmol), and carbon tetrachloride (2.5 mL)was refluxed for 4 h before it was cooled to room temperature andfiltered. The filtrate was washed with water, saturated aqueous sodiumbicarbonate solution, and brine before it was dried over anhydroussodium sulfate and concentrated in vacuo to give the title compound as ayellow solid (128 mg): MS: (+) m/z 374.0, 376.0 (M+1, ⁷⁹Br/⁸¹Br), MS:(−) m/z 372.1, 374.1 (M−1, ⁷⁹Br/⁸¹Br).

g. 1-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acid methylester

A mixture of 1-bromo-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (1.22 g, 3.3 mmol), copper(I) cyanide (585 mg, 6.6 mmol),and anhydrous dimethylformamide (16 mL) was refluxed for ten minutesbefore it was cooled to room temperature and diluted with water. To theresulting slurry was added a chloroform/isopropanol mixture (3:1, 150mL). After stirring for 10 minutes the solid components were separatedby filtration and discarded. The organic layer of the filtrate waswashed with water, and brine before it was dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel with a gradient of methanol anddichloromethane to give the title compound as a white solid (574 mg): ¹HNMR (CDCl₃, 200 MHz): δ=12.26 (s, 1H), 8.15 (m, 1H), 7.71 (t, J=8.2 Hz,1H), 7.38 (m, 2H), 7.17 (m, 4H), 4.12 (s, 3H).

h. [(1-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of 1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (322 mg, 1.0 mmol), glycine (1.51 g, 20.1 mmol), and a 0.5M sodium methoxide solution in methanol (38.2 mL) was refluxed for 31 hbefore it was cooled to room temperature and concentrated in vacuo.Water (75 mL) was added and the pH of the yellow suspension was adjustedto 10 with aqueous 1 N HCl. A clear yellow solution was obtained after 5minutes of sonication. The solution was washed with dichloromethane(2×50 mL). The remaining aqueous layer was acidified to pH=3 withaqueous 1 N HCl. The white precipitate formed was separated byfiltration, washed with water and dried in vacuo to give the titlecompound as a white solid (354 mg): ¹H NMR (DMSO-d₆, 200 MHz): δ=12.86(bs, 1H), 9.56 (t, 1H), 8.09 (m, 1H), 7.88 (t, J=8.2 Hz, 1H), 7.47 (m,2H), 7.21 (m, 4H), 4.05 (d, J=5.8 Hz, 2H).

Example 10{[1-Cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 3-(4-Fluoro-phenoxy)-2-methyl-benzoic acid ethyl ester

A mixture of 3-iodo-2-methyl-benzoic acid ethyl ester (6.29 g, 0.02mol), para-fluorophenol (4.86 g, 0.04 mol), cesium carbonate (14.14 g,0.04 mol), 2,2,6,6-tetramethylheptane-3,5-dione (447 μL, 2 mmol) andcopper (I) chloride (1.07 g, 0.01 mol) in anhydrousN-methyl-2-pyrrolidone (38 mL) was heated at 130° C. for 3 days beforeit was cooled to room temperature, quenched with water and filtered. Thefiltrate was extracted with ethyl acetate. The organic layer was washedtwice with 0.5 N sodium hydroxide, brine, dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate andhexanes to give the title compound as a pale green oil (2.99 g): ¹H NMR(CDCl₃, 200 MHz): δ=7.63 (m, 1H), 7.18 (m, 2H), 6.98 (m, 2H), 6.85 (m,2H), 4.37 (q, J=7.0 Hz, 2H), 2.45 (s, 3H), 1.40 (t, J=7.0 Hz, 3H).

b. 2-Bromomethyl-3-(4-fluoro-phenoxy)-benzoic acid ethyl ester

A mixture of 3-(4-fluoro-phenoxy)-2-methyl-benzoic acid ethyl ester(2.63 g, 9.60 mmol), N-bromosuccinimide (1.79 g, 10.08 mmol) and benzoylperoxide (116 mg, 0.48 mmol) in carbon tetrachloride (35 mL) wasrefluxed for 4 h before it was cooled to room temperature andpartitioned between dichloromethane and water. The organic layer waswashed with saturated aqueous sodium bicarbonate solution, brine, driedover anhydrous sodium sulfate and concentrated in vacuo to give thetitle compound as a yellow oil (3.44 g): ¹H NMR (CDCl₃, 200 MHz): δ=7.67(m, 1H), 7.27 (m, 2H), 7.01 (m, 4H), 5.09 (s, 3H), 4.42 (q, J=7.2 Hz,2H), 1.44 (t, J=7.2 Hz, 3H).

c.3-(4-Fluoro-phenoxy)-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid ethyl ester

A mixture of 2-bromomethyl-3-(4-fluoro-phenoxy)-benzoic acid ethyl ester(3.37 g, 9.57 mmol), (toluene-4-sulfonylamino)-acetic acid methyl ester(2.33 g, 9.57 mmol), sodium iodide (2.15 g, 14.36 mmol) and potassiumcarbonate (1.98 g, 14.36 mmol) in anhydrous dimethylformamide (22 mL)was stirred at room temperature for 24 h before it was quenched withwater, and extracted with ethyl acetate. The organic layer was washedwith water, brine, dried over anhydrous sodium sulfate and concentratedin vacuo. The residue was purified by flash column chromatography onsilica gel with a gradient of ethyl acetate and hexane to give the titlecompound as a yellow oil (2.67 g): MS: (+) m/z 538.13 (M+Na⁺).

d. 8-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid methylester

To a stirred solution of3-(4-fluoro-phenoxy)-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid ethyl ester (2.66 g, 5.17 mmol) in anhydrous methanol (8 mL) wasadded dropwise a solution of sodium methoxide (30% wt, 2.8 mL) andmethanol (2 mL) at 0° C. The mixture was stirred at 0° C. for 10 minutesand then 3 h at room temperature before it was concentrated in vacuo.Water was added and the pH of the slurry was adjusted with 1 N HCl topH=10. The resulting precipitate was collected by filtration, washedwith water, saturated sodium bicarbonate, and water, and was dried invacuo to give the title compound as a white solid (1.51 g): MS: (+) m/z314.07 (M+1).

e. 1-Bromo-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester

A mixture of 8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid methyl ester (1.21 g, 3.87 mmol), N-bromosuccinimide (723 mg, 4.06mmol) and benzoyl peroxide (47 mg, 0.19 mmol) in carbon tetrachloride(20 mL) was refluxed for 4 h before it was cooled to room temperatureand partitioned between dichloromethane and water. The organic layer waswashed with saturated aqueous sodium bicarbonate, brine, and was driedover anhydrous sodium sulfate and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel with a gradient ofdichloromethane and hexane to give the title compound as a pale yellowsolid (843 mg): MS: (+) m/z 392.00, 393.93 (M+1, ⁷⁹Br/⁸¹Br).

f. 1-Cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester

A mixture of1-bromo-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester (400 mg, 1.02 mmol) and copper(I) cyanide (183 mg, 2.04mmol) in anhydrous dimethylformamide (4 mL) was refluxed for ten minutesbefore it was cooled to room temperature and quenched with water. Theslurry was stirred with chloroform/isopropanol (3:1, 70 mL) and waterfor ten minutes and filtered. The organic layer was washed with water,brine, dried over anhydrous sodium sulfate, concentrated in vacuo, andpurified by flash column chromatography on silica gel with a gradient ofmethanol and dichloromethane to give the title compound as a white solid(158 mg): MS: (+) m/z 339.07 (M+1).

g.{[1-Cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester (112 mg, 0.33 mmol), glycine (496 mg, 6.6 mmol), and a 0.5M solution of sodium methoxide in methanol (12.5 mL, 6.25 mmol) wasrefluxed for 39 h before it was cooled to room temperature andconcentrated in vacuo. Water (75 mL) was added and the pH of the yellowsuspension was adjusted to pH=10 with 1 N HCl. The suspension was washedwith methylene chloride (2×50 mL). The remaining aqueous layer wasacidified to pH=3 with 1 N HCl. The resulting white precipitate wascollected by filtration, washed with water, and dried in vacuo to givethe title compound as a white solid (109 mg): ¹H NMR (DMSO-d₆, 200 MHz):δ=12.85 (bs, 1H), 9.57 (br s, 1H), 8.09 (d, 1H), 7.83 (t, 1H), 7.27 (m,5H), 4.05 (d, J=5.8 Hz, 2H); MS: (+) m/z 382.00 (M+1).

Example 11[(1-Cyano-4-hydroxy-6-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Bromo-4-hydroxy-6-methoxy-isoquinoline-3-carboxylic acid butylester

A mixture of 1,4-dihydroxy-6-methoxy-isoquinoline-3-carboxylic acidbutyl ester (1.53 g, 5.26 mmol, prepared according to US 2004/0254215 A1or scheme 2), phosphorus oxybromide (6.04 g, 21.05 mmol), andacetonitrile (40 mL) was refluxed for 16 h before it was cooled to roomtemperature and partitioned between dichloromethane and aqueoussaturated sodium bicarbonate solution for twenty minutes. The mixturewas then filtered. The organic layer of the filtrate was separated,washed with water, and brine, dried over anhydrous sodium sulfate andwas concentrated in vacuo. The residue was purified by flash columnchromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a yellow solid (290 mg):¹H NMR (CDCl₃, 200 MHz): δ=11.84 (s, 1H), 8.14 (d, J=9.4 Hz, 1H), 7.62(d, J=2.3 Hz, 1H), 7.83 (dd, J=8.0 Hz, 2.7 Hz, 1H), 4.48 (t, J=7.0 Hz,2H), 1.86 (m, 1H), 1.55 (m, 1H), 0.99 (t, J=7.4 Hz).

b. 1-Cyano-4-hydroxy-6-methoxy-isoquinoline-3-carboxylic acid butylester

A mixture of 1-bromo-4-hydroxy-6-methoxy-isoquinoline-3-carboxylic acidbutyl ester (290 mg, 0.82 mmol) and copper(I) cyanide (147 mg, 1.64mmol) in anhydrous dimethylformamide (4 mL) was refluxed for ten minutesbefore it was cooled to room temperature and quenched with water. Theresulting slurry was stirred with chloroform/isopropanol (3:1, 40 mL)and water for ten minutes and was then filtered. The organic layer ofthe filtrate was separated, washed with water, and brine, before it wasdried over anhydrous sodium sulfate and concentrated in vacuo. Theresidue was purified by flash column chromatography on silica gel with agradient of methanol and dichloromethane to give the title compound as awhite solid (120 mg): MS: (+) m/z 301.01 (M+1).

c. [(1-Cyano-4-hydroxy-6-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of 1-cyano-4-hydroxy-6-methoxy-isoquinoline-3-carboxylic acidbutyl ester (104 mg, 0.35 mmol), glycine (523 mg, 6.97 mmol), and a 0.5M solution of sodium methoxide in methanol (13.2 mL, 6.6 mmol) wasrefluxed for 3 days before it was cooled to room temperature andconcentrated in vacuo. The residue was dissolved in water (30 mL) andthe solution was extracted with methyl tert-butyl ether (2×25 mL). Theremaining aqueous layer was acidified to pH=3 with 1N HCl (10 mL). Theresulting white precipitate was collected by filtration, washed withwater and dried in vacuo to give the title compound as a white solid (82mg): ¹H NMR (DMSO-d₆, 200 MHz): δ=9.53 (t, 1H), 8.10 (d, J=9.0 Hz, 1H),7.57 (m, 2H), 4.02 (d, J=6.2 Hz, 2H), 3.98 (s, 3H); MS: (+) m/z 302.00(M+1).

Example 12[(1-Cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butylester

Synthesized from 1-bromo-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylicacid butyl ester (prepared according to US 2004/0254215 A1 or scheme 2,¹H NMR (CDCl₃): δ=11.76 (s, 1H), 8.22 (d, 1H), 7.68 (d, 1H), 7.10 to7.55 (m, 6H), 4.46 (t, 2H), 1.85 (m, 2H), 1.48 (m, 2H), 0.99 (t, 3H))and CuCN in analogy to Example 3a.; MS-(−)-ion: M−1=361.3.

b. [(1-Cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was obtained from1-cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carboxylic acid butyl esterand glycine in analogy to Example 1b.; MS-(−)-ion: M−1=362.1.

Example 13{[1-Cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 1-Bromo-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of6-(4-fluoro-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (7.43 g, 20 mmol, prepared according to US 2004/0254215 A1 orscheme), POBr₃ (17.38 g, 60 mmol), and anhydrous acetonitrile (140 mL)was refluxed with stirring for 60 minutes before it was concentrated invacuo. To the residue was added ethyl acetate (400 mL), NaHCO₃ (60 g),and then water in small portions (400 mL) with stirring. After stirringfor 30 min at room temperature the mixture was filtered through a pad ofcelite. The organic phase of the filtrate was separated, and dried overMgSO₄. Then silica gel was added and the mixture was concentrated invacuo. The residue was added on top of a short column filled with silicagel. Elution using CH₂Cl₂ as the solvent gave the title compound as anoff-white solid (930 mg); ¹H NMR (CDCl₃, 200 MHz): δ=11.76 (s, 1H), 8.22(d, J=9.4 Hz, 1H), 7.61 (d, J=2.3 Hz, 1H), 7.50 (dd, J=9.0, 2.3 Hz, 1H),7.10 to 7.13 (m, 4H), 4.46 (t, J=7.0 Hz, 2H), 1.85 (m, 2H), 1.45 (m,2H), 0.99 (t, J=7.4 Hz, 3H).

b. 1-Cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Synthesized from1-bromo-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester and CuCN in analogy to Example 3a.; MS-(−)-ion: M−1=379.2.

c.{[1-Cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was obtained from1-cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester and glycine in analogy to Example 1b.; MS-(−)-ion:M−1=380.0.

Example 14{[1-Cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a.1-Bromo-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester

To mixture of 4-Hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester (1.837 g, 5 mmol; for preparation, see example 28b) andN-bromosuccinimide (1.079 g, 6 mmol) was added anhydrous MeCN withstirring. After ca. 10 min another portion of MeCN (3 mL) was added andstirring continued for 20 min. The mixture was then partitioned betweenwater (100 mL) and CCl₄ (100 mL). The organic phase was separated, driedover MgSO₄ and concentrated in vacuo. The residue was recrystallizedfrom EtOAc to give the title compound as white needles (1.345 g);MS-(+)-ion: M+1, ⁷⁹Br/⁸¹Br=445.8 and 447.8.

b. 1-Cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-Bromo-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester and CuCN in analogy to example 3a; MS-(+)-ion: M+1=392.9.

c.{[1-Cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-Cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester and glycine in analogy to example 1b; MS-(−)-ion: M−1=391.9.

Example 15[(1-Cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-Bromo-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (prepared as shown in Scheme 2, according to US 2004/0254215 A1)and CuCN in analogy to example 3a; MS-(+)-ion: M+1=378.9.

b.[(1-Cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was synthesized from1-Cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester and glycine in analogy to example 1b; MS-(−)-ion: M−1=377.9.

Example 16[(1-Cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid a. 1-Cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-Bromo-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester (prepared as shown in Scheme 2, according to US 2004/0254215 A1)and CuCN in analogy to example 3a; MS-(+)-ion: M+1=378.9.

b.[(1-Cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was synthesized from1-Cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acid butylester and glycine in analogy to example 1b; MS-(−)-ion: M−1=378.0.

Example 17{[1-Cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-(2,6-Dimethyl-phenoxy)-phthalonitrile

A mixture of 4-nitro-phthalonitrile (1 eq), 2,6-dimethylphenol (1.2 eq),K₂CO₃ (2 eq), and DMF (1 mL/mmol 4-nitro-phthalonitrile) was heatedunder nitrogen at 60° C. for 3 h with stirring. After cooling to ambienttemperature the mixture was poured into water (6 mL/mL DMF) withstirring. The mixture was extracted twice with EtOAc. The combinedorganic phases were dried over MgSO₄ and concentrated in vacuo. Theresidue was recrystallized from EtOH to give the title compound as a tansolid (yield: 87%); ¹H NMR (CDCl₃, 200 MHz): δ=7.70 (d, 1H), 7.05 to7.16 (m, 5H), 2.08 (s, 6H).

b. 4-(2,6-Dimethyl-phenoxy)-phthalic acid

A mixture of 4-(2,6-dimethyl-phenoxy)-phthalonitrile, aqueous KOH (45 wt% KOH; 0.5 mL/mmol), and MeOH (0.5 mL/mmol) was refluxed for 4 days withstirring before it was diluted with water (5 mL/mmol) and acidified byaddition of concentrated hydrochloric acid. The resulting mixture wasextracted with EtOAc. The organic phase was dried over MgSO₄ andconcentrated in vacuo to give the title compound as a tan solid (yield:99%); MS-(−)-ion: M−1=285.5.

c. [5-(2,6-Dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid

4-(2,6-Dimethyl-phenoxy)-phthalic acid and an equimolar amount ofglycine were ground thoroughly together in a mortar. The mixture wasthen heated at 220 to 240° C. in an oil-bath in vacuo until theformation of water ceased (ca. 30 min) to give the title compound as adark glass (yield: 99%); MS-(−)-ion: M−1=324.5.

d. [5-(2,6-Dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

To a solution of[5-(2,6-Dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid in MeOH (1 mL/mmol) was added concentrated sulfuric acid (35μl/mmol) and the mixture was refluxed with stirring for 16 h before itwas diluted with water (6.5 mL/mmol) and extracted twice with EtOAc. Thecombined organic phases were washed with saturated NaHCO₃ solution,dried over MgSO₄, and concentrated in vacuo to give the title compoundas a tan solid (yield: 96%); MS-(+)-ion: M+1=340.5.

e. 6-(2,6-Dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester

Sodium (1 eq) was dissolved in n-butanol (1.6 mL/mmol) with stirring at70° C. Subsequently, the temperature was raised to 95° C. before asolution of[5-(2,6-Dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester (0.5 eq) in hot n-butanol (2.3 mL/mmol) was added inone portion with stirring. Stirring was continued at 95° C. for 3 hbefore the mixture was concentrated in vacuo. To the residue was added 2N hydrochloric acid (1.3 eq) and EtOAc (ca. 4-fold volume) and themixture was stirred vigorously for 45 min. Subsequently, the solidcomponent was sucked off, washed with water and dried in vacuo before itwas suspended in EtOAc (ca. 20 mL/g) and the mixture was refluxed withstirring for 2 h. After cooling to ambient temperature the solidcomponent was sucked off, washed with EtOAc and dried in vacuo to givethe title compound as an off-white solid (yield: 43%); MS-(+)-ion:M+1=382.5

f. 1-Bromo-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of6-(2,6-Dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acidbutyl ester (4 mmol, 1.53 g), POBr₃ (16 mmol, 4.63 g), MeCN (30 mL) wasrefluxed gently with stirring before it was concentrated in vacuo. Theresidue was dissolved in CH₂Cl₂ (100 mL). To the solution was addedNaHCO₃ (20 g) and then water (100 mL) in small portions with stirring.The mixture was stirred for 1 h at ambient temperature before theorganic phase was separated and dried over MgSO₄. Concentration andpurification of the residue by flash column chromatography on silica gelusing CH₂Cl₂ as the eluent gave the title compound as a yellowish oil(640 mg); MS-(+)-ion: M+1, ⁷⁹Br/⁸¹Br=443.9 and 445.9.

g. 1-Cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-Bromo-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and CuCN in analogy to example 3a; MS-(+)-ion:M+1=390.9.

h.{[1-Cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-Cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=390.0.

Example 18[(1-Cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid a. 2-Methyl-6-phenoxy-benzoic acid

2-Chloro-6-methyl-benzoic acid (30 mmol, 5.22 g) and phenol (40 mmol,3.8 g) were dissolved in a solution of NaOMe (30 wt %) in MeOH (ca. 66mmol, 12 mL). To the solution was added copper bronze (3 mmol, 193 mg)before it was concentrated in vacuo. Then, 1,2-dichlorobenzene (24 mL)was added and the mixture was refluxed under nitrogen for 2 h withstirring. After cooling to ambient temperature water (200 mL) and Et₂O(150 mL) were added and the mixture was stirred vigorously for 30 minbefore the organic phase was separated and discarded. The aqueous phasewas washed with Et₂O (150 mL) before it was acidified by addition of 5 Nhydrochloric acid. The resulting mixture was extracted with ethylacetate (1×100 mL). The organic phase was dried over MgSO₄ andconcentrated in vacuo. The residue was recrystallized fromhexanes/toluene to give the title compound as a tan solid (4.55 g);MS-(−)-ion: M−1=226.8.

b. 2-Methyl-6-phenoxy-benzoic acid methyl ester

A mixture of 2-methyl-6-phenoxy-benzoic acid (19.9 mmol, 4.54 g),methanol (20 mL), and concentrated sulfuric acid (1.5 mL) was refluxedwith stirring for 18 h before it was concentrated in vacuo. To theresidue was added water (50 mL) and the mixture was neutralized byadding small portions of NaHCO₃ with stirring. Subsequently, the mixturewas extracted with ethyl acetate (1×50 mL). The organic phase was driedover MgSO₄ and concentrated in vacuo. Purification of the residue byflash column chromatography on silica gel using hexanes:ethylacetate=7:3 as the eluent gave the title compound as a yellowish oil(2.16 g); MS-(+)-ion: M+1=242.8.

c.2-{[Methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-6-phenoxy-benzoicacid methyl ester

A mixture of 2-methyl-6-phenoxy-benzoic acid methyl ester (8.9 mmol,2.15 g), N-bromosuccinimide (9.1 mmol, 1.64 g), benzoyl peroxide (0.44mmol, 110 mg), and CCl₄ (35 mL) was refluxed with stirring for 6 h.After cooling to ambient temperature the mixture was filtered and thefiltrate was concentrated in vacuo to give a yellowish oil (3.01 g). Theoil (3.00 g) was dissolved in dry DMF (8 mL). NaI (2.42 g), K₂CO₃ (2.21g), and (toluene-4-sulfonylamino)-acetic acid methyl ester (2.04 g) wereadded and the mixture was stirred at ambient temperature for 18 h beforewater was added with stirring. The aqueous phase was then decanted fromthe oily precipitate formed. The oil was dissolved in ethyl acetate (70mL) and the solution was washed with concentrated aqueous NaHCO₃solution before it was dried over MgSO₄ and concentrated in vacuo togive the title compound as a dark oil (3.87 g). The crude product wasused in the next step without further purification; MS-(+)-ion:M+23=506.0.

d. 4-Hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methyl ester

To a solution of crude2-{[Methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-6-phenoxy-benzoicacid methyl ester (3.87 g) in methanol (24 mL) was added a solution ofNaOMe (30 wt %) in methanol (4 mL) with stirring. After stirring for 3days at ambient temperature the mixture was concentrated in vacuo. Tothe residue was added 1 N hydrochloric acid (20 mL) and the mixture wasextracted with ethyl acetate (1×100 mL). The organic phase was thenwashed with concentrated aqueous NaHCO₃ solution (3×100 mL), dried overMgSO₄ and concentrated in vacuo. The residue was recrystallized fromethyl acetate to give the title compound as a yellowish solid (630 mg);MS-(+)-ion: M+1=295.8.

e. 1-Bromo-4-hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methylester

A mixture of 4-hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methylester (1.55 mmol, 458 mg), N-bromosuccinimide (1.7 mmol, 306 mg),benzoyl peroxide (0.08 mmol, 19 mg), and CCl₄ (10 mL) was refluxed withstirring for 2 h. After cooling to ambient temperature the mixture wasfiltered and the filtrate was concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel usinghexanes:ethyl acetate=9:1 as the eluent to give the title compound as anoff-white solid (225 mg); MS-(+)-ion: M+1, ⁷⁹Br/⁸¹Br=373.9 and 375.8.

f. 1-Cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methylester

The title compound was synthesized from1-Bromo-4-hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methyl esterand CuCN in analogy to example 3a; MS-(+)-ion: M+1=320.8.

g. [(1-Cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was synthesized from1-Cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carboxylic acid methyl esterand glycine in analogy to example 1b; MS-(+)-ion: M+1=363.9.

Example 19{[1-Cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a. 3-Iodo-2-methyl-benzoic acid methyl ester

A mixture of 3-iodo-2-methyl-benzoic acid (90 mmol, 23.6 g), methanol(250 mL), and concentrated sulfuric acid (13 mL) was refluxed withstirring for 40 h before it was concentrated in vacuo. The residue wasdissolved in ethyl acetate and the mixture was neutralized by addingsmall portions of a saturated aqueous NaHCO₃ solution with stirring. Theorganic phase was dried over MgSO₄ and concentrated in vacuo to give thetitle compound as a yellowish oil (24.3 g); ¹H NMR (CDCl₃, 200 MHz):δ=7.96 (d, 1H), 7.71 (d, 1H), 6.91 (t, 1H), 3.89 (s, 3H), 2.66 (s, 3H).

b.3-Iodo-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester

A mixture of 3-iodo-2-methyl-benzoic acid methyl ester (75 mmol, 20.7g), N-bromosuccinimide (76.6 mmol, 13.8 g), benzoyl peroxide (890 mg),and CCl₄ (300 mL) was refluxed with stirring for 15 h. After cooling toambient temperature the mixture was filtered and the filtrate wasconcentrated in vacuo to give the title compound as a tan oil. The oil(26.3 g) was dissolved in dry DMF (75 mL). NaI (22.4 g), K₂CO₃ (20.5 g),and (toluene-4-sulfonylamino)-acetic acid methyl ester (19 g) were addedand the mixture was stirred at ambient temperature for 24 h before itwas poured into water (900 mL). The mixture was extracted with ethylacetate (2×250 mL). The combined organic phases were washed with asolution of sodium meta-bisulfite (20 g) in water (300 mL) and water(2×300 mL) before they were dried over MgSO₄ and concentrated in vacuoto give the title compound as a dark gum (38.1 g). The crude product wasused in the next step without further purification; MS-(+)-ion:M+23=539.9.

c. 4-Hydroxy-8-iodo-isoquinoline-3-carboxylic acid methyl ester

To a solution of crude3-Iodo-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester (37.8 g) in methanol (220 mL) was added a solution ofNaOMe (30 wt %) in methanol (40 mL) with stirring. After stirring for 18h at ambient temperature the mixture was concentrated in vacuo. To theresidue was added 1 N hydrochloric acid (200 mL) and the mixture wasextracted with hot ethyl acetate (1×300 mL). The organic phase was thenwashed with concentrated aqueous NaHCO₃ solution (3×200 mL), dried overMgSO₄ and concentrated in vacuo. The residue was recrystallized fromethyl acetate to give the title compound as a tan solid (10.1 g);MS-(+)-ion: M+1=329.8.

d. 4-Hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acid methylester

A mixture of 4-Hydroxy-8-iodo-isoquinoline-3-carboxylic acid methylester (7 mmol, 2.3 g), 4-methoxy-phenol (35 mmol, 4.39 g), Cs₂CO₃ (35mmol, 11.42 g), 2,2,6,6-tetramethyl-heptane-3,5-dione (2.8 mmol, 0.59mL), CuCl (7 mmol, 0.70 g), and anhydrous DMF (42 mL) was refluxed undernitrogen with stirring for 15 min. before it was poured into ethylacetate (700 mL). Water (700 mL) and 5 N hydrochloric acid (5 mL) wereadded and the mixture was stirred for 15 min. The organic phase was thenseparated and washed with water (2×700 mL) before it was dried overMgSO₄ and concentrated in vacuo. Purification of the residue by flashcolumn chromatography on silica gel using hexanes:EtOAc=75:25 as theeluent gave the title compound as a tan solid (234 mg); MS-(+)-ion:M+1=326.4.

e. 1-Bromo-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester

A mixture of 4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester (1.17 mmol, 381 mg), N-bromosuccinimide (1.3 mmol, 234mg), benzoyl peroxide (0.06 mmol, 15 mg), and CCl₄ (8 mL) was refluxedwith stirring for 2.5 h. After cooling to ambient temperature silica gelwas added and the mixture was concentrated in vacuo. The residue wasadded on top of a chromatography column filled with silica gel. Elutionwith CH₂Cl₂ gave the title compound as a tan solid (403 mg); MS-(+)-ion:M+1, ⁷⁹Br/⁸¹Br=404.3 and 406.3.

f. 1-Cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester

The title compound was synthesized from1-bromo-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester and CuCN in analogy to example 3a; MS-(+)-ion: M+1=351.4.

g.{[1-Cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=392.4.

Example 20{[1-Cyano-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-Hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester

The title compound was synthesized from4-hydroxy-8-iodo-isoquinoline-3-carboxylic acid methyl ester and3-methoxy-phenol in analogy to example 19d; MS-(+)-ion: M+1=326.4.

b. 1-Bromo-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester

A mixture of 4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester (1.75 mmol, 569 mg), N-bromosuccinimide (2 mmol, 360mg), benzoyl peroxide (0.09 mmol, 22 mg), and CCl₄ (12 mL) was refluxedwith stirring for 4 h. After cooling to ambient temperature the mixturewas filtered, silica gel was added to the filtrate and the mixture wasconcentrated in vacuo. The residue was added on top of a chromatographycolumn filled with silica gel. Elution with CH₂Cl₂ gave a yellowishsolid (435 mg). A mixture of this solid (283 mg), CuCN (127 mg), andanhydrous DMF (2.8 mL) was refluxed with stirring under nitrogen for 15min. After cooling to ambient temperature the mixture was diluted withethyl acetate (200 mL). The resulting mixture was stirred for 15 min andthen filtered through a pad of celite. The filtrate was washed with 0.1Nhydrochloric acid (1×300 mL) and water (2×300 mL), and dried over MgSO₄.Subsequently, the mixture was concentrated in vacuo to give a tan solid(178 mg). A mixture of this solid (175 mg), sodium acetate (49 mg), Pd/C(10 wt % Pd, 50 wt % water; 100 mg), methanol (10 mL), and ethyl acetate(20 mL) was stirred under a hydrogen atmosphere (ambient pressure) for18 h before it was filtered through a pad of celite. The filtrate wasconcentrated in vacuo. To the residue was added saturated aqueous NaHCO₃solution (20 mL) and the mixture was extracted with ethyl acetate (1×40mL). The organic phase was dried over MgSO₄ and concentrated in vacuo.Purification of the residue by flash column chromatography on silica gelusing CH₂Cl₂:ethyl acetate=98:2 as the eluent gave the title compound asan off-white solid (101 mg); MS-(−)-ion: M−1=349.4.

c.{[1-Cyano-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-cyano-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=392.4.

Example 21{[1-Cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-Hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester

The title compound was synthesized from4-hydroxy-8-iodo-isoquinoline-3-carboxylic acid methyl ester and2-methoxy-phenol in analogy to example 19d; MS-(+)-ion: M+1=326.4.

b. 1-Bromo-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester

The title compound was synthesized from4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acid methylester and N-bromosuccinimide in analogy to example 19e; MS-(+)-ion: M+1,⁷⁹Br/⁸¹Br=404.3 and 406.3.

c. 1-Cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylicacid methyl ester

The title compound was synthesized from1-bromo-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester and CuCN in analogy to example 3a; MS-(+)-ion: M+1=351.4.

d.{[1-Cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carboxylic acidmethyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=392.5.

Example 22[(7-Benzyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida. 5-Benzyl-3H-isobenzofuran-1-one

A mixture of 5-bromo-3H-isobenzofuran-1-one (14 mmol, 3.04 g),benzylzinc bromide solution (0.5 M in THF, 28 mmol, 56 mL),[1,1′bis(diphenylphosphino)-ferrocene]dichloropalladium(II) 1:1 complexwith CH₂Cl₂ (0.07 mmol, 57 mg), and anhydrous 1,4-dioxane (70 mL) wasrefluxed with stirring under nitrogen for 40 h. After cooling to ambienttemperature silica gel was added and the mixture was concentrated invacuo. The residue was added on top of a chromatography column filledwith silica gel. Elution with hexanes:ethyl acetate=9:1 gave a yellowsolid. Further purification by recrystallization from ethylacetate/hexanes gave the title compound (1.37 g) as white needles; ¹HNMR (CDCl₃, 200 MHz): δ=7.83 (d, 1H), 7.16 to 7.39 (m, 7H), 5.26 (s,2H), 4.11 (s, 2H).

b.4-Benzyl-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester

A mixture of 5-benzyl-3H-isobenzofuran-1-one (6 mmol, 1.35 g), boricacid (0.18 mmol, 11 mg), triphenylphosphine oxide (0.18 mmol, 51 mg),and thionyl chloride (7.8 mmol, 0.59 mL) was refluxed in an oil bath(bath temperature 130 to 140° C.) for 18 h with stirring. Subsequently,methanol (6 mL) was added and the mixture was stirred for 15 min beforeit was concentrated in vacuo. The residue was dissolved in ethyl acetate(40 mL). The solution was washed with saturated aqueous NaHCO₃ solution(2×20 mL), dried over MgSO₄ and was concentrated in vacuo to give ayellowish oil (1.51 g). The oil (906 mg) was dissolved in anhydrous DMF(5 mL). NaI (1.0 g), K₂CO₃ (912 mg), and(toluene-4-sulfonylamino)-acetic acid methyl ester (803 mg) were addedand the mixture was stirred at ambient temperature for 15 h before itwas poured into water (50 mL). Traces of iodine were removed by adding asmall amount of sodium meta-bisulfite before extracting the mixture withethyl acetate (1×50 mL). The organic phase was then washed with water(1×50 mL), dried over MgSO₄ and concentrated in vacuo to give the crudetitle compound as a dark gum (1.58 g) that was used in the next stepwithout further purification; MS-(+)-ion: M+1=481.8.

c. 7-Benzyl-4-hydroxy-isoquinoline-3-carboxylic acid methyl ester

To a solution of crude4-benzyl-2-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester (1.54 g) in methanol (1.75 mL) was added a solution ofNaOMe (30 wt %) in methanol (1.75 mL) with stirring. After stirring for5 h at ambient temperature the mixture was concentrated in vacuo andwater (20 mL) was added. The pH of the mixture was adjusted to 7 to 8 byaddition of 6N hydrochloric acid before the mixture was extracted withethyl acetate (2×25 mL). The combined organic phases were then driedover MgSO₄ and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel using hexanes:ethyl acetate=7:3 asthe eluent to give the title compound as an off-white solid (451 mg);MS-(+)-ion: M+1=294.0.

d. 7-Benzyl-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid methylester

A mixture of 7-benzyl-4-hydroxy-isoquinoline-3-carboxylic acid methylester (1 mmol, 293 mg), N-bromosuccinimide (1.2 mmol, 214 mg) andanhydrous MeCN (10 mL) was stirred at ambient temperature for 4 daysbefore silica gel was added and the mixture was concentrated in vacuo.The residue was added on top of a chromatography column filled withsilica gel. Elution with hexanes:ethyl acetate=75:25 gave the titlecompound as an off-white solid (48 mg); MS-(+)-ion: M+1, ⁷⁹Br/⁸¹Br=372.4and 374.4.

e. 7-Benzyl-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid methylester

The title compound was synthesized from7-benzyl-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid methyl esterand CuCN in analogy to example 3a; MS-(−)-ion: M−1=317.4.

f. [(7-Benzyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

The title compound was synthesized from7-benzyl-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid methyl esterand glycine in analogy to example 1b; MS-(−)-ion: M−1=360.5.

Example 23{[1-Cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 2-(4-Fluoro-phenoxy)-6-methyl-benzoic acid

The title compound was synthesized from 2-chloro-6-methyl-benzoic acidand 4-fluorophenol in analogy to example 18a; MS-(−)-ion: M−1=245.5.

b. 2-(4-Fluoro-phenoxy)-6-methyl-benzoic acid methyl ester

A mixture of 2-(4-fluoro-phenoxy)-6-methyl-benzoic acid (21.6 mmol, 5.32g), dimethylsulfate (43.2 mmol, 4.2 mL), K₂CO₃ (43.2 mmol, 6 g), anddiethyl ketone (80 mL) was refluxed with stirring for 18 h before it wasconcentrated in vacuo. To the residue was added water (50 mL) and themixture was extracted with ethyl acetate (1×100 mL). The organic phasewas dried over MgSO₄ and concentrated in vacuo to give the titlecompound as a tan oil (5.5 g); ¹H NMR (CDCl₃, 200 MHz): δ=7.20 (t, 1H),6.92 to 7.04 (m, 5H), 6.66 (d, 1H), 3.84 (s, 3H), 2.35 (s, 3H).

c.2-(4-Fluoro-phenoxy)-6-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester

The crude title compound was synthesized from2-(4-fluoro-phenoxy)-6-methyl-benzoic acid methyl ester,N-bromosuccinimide, and (toluene-4-sulfonylamino)-acetic acid methylester in analogy to example 18c and was used in the following stepwithout further purification; MS-(+)-ion: M+23=524.4.

d. 5-(4-Fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid methylester

The title compound was synthesized from crude2-(4-fluoro-phenoxy)-6-{[methoxycarbonylmethyl-(toluene-4-sulfonyl)-amino]-methyl}-benzoicacid methyl ester in analogy to example 18d; MS-(+)-ion: M+1=314.4.

e. 1-Bromo-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester

The title compound was synthesized from5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid methylester and N-bromosuccinimide in analogy to example 18e; MS-(+)-ion: M+1,⁷⁹Br/⁸¹Br=392.4 and 394.3.

f. 1-Cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester

The title compound was synthesized from1-bromo-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester and CuCN in analogy to example 3a; MS-(−)-ion: M−1=337.4.

g.{[1-Cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acidmethyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=380.4.

Example 24{[1-Cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a.1-Cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Synthesized from1-bromo-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (for synthesis, see US 2004/0254215 A1) and CuCN inanalogy to Example 3a; MS-(−)-ion: M−1=389.5.

b.{[1-Cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was obtained from1-cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine in analogy to Example 1b; MS-(−)-ion:M−1=390.5.

Example 25{[1-Cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-(2-Ethyl-6-methyl-phenoxy)-phthalonitrile

The title compound was obtained from 4-nitro-phthalonitrile and2-ethyl-6-methyl-phenol in analogy to example 17a; MS-(+)-ion:M+1=263.5.

b. 4-(2-Ethyl-6-methyl-phenoxy)-phthalic acid

The title compound was obtained from4-(2-Ethyl-6-methyl-phenoxy)-phthalonitrile in analogy to example 17b;MS-(−)-ion: M−1=299.4.

c.[5-(2-Ethyl-6-methyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid

The title compound was obtained from4-(2-Ethyl-6-methyl-phenoxy)-phthalic acid and glycine in analogy toexample 17c; MS-(−)-ion: M−1=338.4.

d.[5-(2-Ethyl-6-methyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

The title compound was obtained from[5-(2-Ethyl-6-methyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid and methanol in analogy to example 17d; MS-(+)-ion: M+1=354.4.

e. 6-(2-Ethyl-6-methyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

To a solution of[5-(2-Ethyl-6-methyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester (22 mmol, 7.77 g) in anhydrous n-butanol (150 mL) wasadded a 1 N solution of sodium in n-butanol (45 mmol, 45 mL) at 95° C.in one portion with stirring. Stirring was continued at 95° C. for 3 hbefore the mixture was concentrated in vacuo. To the residue was added 2N hydrochloric acid (60 mmol, 30 mL) and EtOAc (150 mL) and the mixturewas stirred vigorously for 45 min. Subsequently, the solid component wassucked off, washed with water and dried in vacuo to give the titlecompound as a tan solid (623 mg); MS-(+)-ion: M+1=396.5.

f.1-Bromo-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of6-(2-Ethyl-6-methyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester (2.83 mmol, 1.12 g), POBr₃ (11 mmol, 3.19 g), MeCN (20mL) was refluxed gently with stirring for 1 h before it was concentratedin vacuo. The residue was dissolved in CHCl₃ (50 mL). Water (10 mL) andsubsequently small portions of NaHCO₃ (6 g) were added with stirring.The mixture was stirred for 30 min at ambient temperature before it wasfiltered through a pad of celite. The filtrate was dried over MgSO₄.Concentration and purification of the residue by flash columnchromatography on silica gel using CH₂Cl₂ as the eluent gave the titlecompound as a tan oil (450 mg); MS-(+)-ion: M+1, ⁷⁹Br/⁸¹Br=458.4 and460.6.

g.1-Cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-Bromo-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and CuCN in analogy to example 3a; MS-(−)-ion:M−1=403.5.

h.{[1-Cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-Cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=404.4.

Example 26{[1-Cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-(2,4,6-Trimethyl-phenoxy)-phthalonitrile

The title compound was obtained from 4-nitro-phthalonitrile and2,4,6-trimethylphenol in analogy to example 17a; MS-(−)-ion: M−1=261.5.

b. 4-(2,4,6-Trimethyl-phenoxy)-phthalic acid

The title compound was obtained from4-(2,4,6-trimethyl-phenoxy)-phthalonitrile in analogy to example 17b;MS-(−)-ion: M−1=299.4.

c.[1,3-Dioxo-5-(2,4,6-trimethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid

The title compound was obtained from4-(2,4,6-trimethyl-phenoxy)-phthalic acid and glycine in analogy toexample 17c; MS-(−)-ion: M−1=338.4.

d.[1,3-Dioxo-5-(2,4,6-trimethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

The title compound was obtained from[1,3-dioxo-5-(2,4,6-trimethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid and methanol in analogy to example 17d; MS-(+)-ion: M+1=354.4.

e. 1,4-Dihydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

The title compound was obtained from[1,3-dioxo-5-(2,4,6-trimethyl-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester in analogy to example 25e; MS-(+)-ion: M+1=396.4.

f.1-Bromo-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

The title compound was obtained from1,4-dihydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester and POBr₃ in analogy to example 25f; MS-(+)-ion: M+1,⁷⁹Br/⁸¹Br=458.4 and 460.4.

g.1-Cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-bromo-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester and CuCN in analogy to example 3a; MS-(−)-ion:M−1=403.4.

h.{[1-Cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from1-cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carboxylicacid butyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=404.4.

Example 27{[6-(4-Chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a. 4-(4-Chloro-2,6-dimethyl-phenoxy)-phthalonitrile

The title compound was obtained from 4-nitro-phthalonitrile and4-chloro-2,6-dimethyl-phenol in analogy to example 17a; MS-(+)-ion:M+1=283.4.

b. 4-(4-Chloro-2,6-dimethyl-phenoxy)-phthalic acid

The title compound was obtained from4-(4-chloro-2,6-dimethyl-phenoxy)-phthalonitrile in analogy to example17b; MS-(−)-ion: M−1=319.4.

c.[5-(4-Chloro-2,6-dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid

The title compound was obtained from4-(4-chloro-2,6-dimethyl-phenoxy)-phthalic acid and glycine in analogyto example 17c; MS-(−)-ion: M−1=358.4.

d.[5-(4-Chloro-2,6-dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester

The title compound was obtained from[5-(4-chloro-2,6-dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid and methanol in analogy to example 17d; MS-(−)-ion: M−1=372.4.

e.6-(4-Chloro-2,6-dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

The title compound was obtained from[5-(4-chloro-2,6-dimethyl-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid methyl ester in analogy to example 25e; MS-(+)-ion: M+1=416.4.

f.1-Bromo-6-(4-chloro-2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

The title compound was obtained from6-(4-chloro-2,6-dimethyl-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester and POBr₃ in analogy to example 25f; MS-(+)-ion: M+1,⁷⁹Br/⁸¹Br=478.3 and 480.3.

g.6-(4-Chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

The title compound was synthesized from1-bromo-6-(4-chloro-2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and CuCN in analogy to example 3a; MS-(−)-ion:M−1=423.4.

h.{[6-(4-Chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

The title compound was synthesized from6-(4-Chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine in analogy to example 1b; MS-(−)-ion:M−1=424.3.

Example 28{[1-Cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 6- and7-(4-Methoxy-phenoxy)-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

POCl₃ (1.2 g, 7.8 mmol) was added to a mixture of 6- and7-(4-methoxy-phenoxy)-1,4-dihydroxy-isoquinoline-3-carboxylic acid butylester (see U.S. Patent Application Publication No. 2004/0254215) (3.0 g,7.8 mmol) in anhydrous toluene (40 mL). Resulting mixture was microwavedat 130° C. for 15 min (ramp time 20 min). Reaction mixture wasconcentrated and carefully quenched with saturated NaHCO₃ solution (150mL). After stirred at room temperature for 10 min, it was extracted withEtOAc (2×200 mL). Combined organic layers were washed with water andbrine, dried over MgSO₄, filtered, and concentrated to give a mixture ofthe title compounds (2.4 g). MS-(+)-ion: M+1=402.25.

b) 7-(4-Methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester

To a solution of a 6- and7-(4-methoxy-phenoxy)-1-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester mixture (10 g, 24.9 mmol) in EtOAc (160 mL) was added 10%Pd/C (50% wet) (3.7 g) and then ammonium formate (15.7 g, 249 mmol).Resulting mixture was refluxed for 4 h. After cooled, it was dilutedwith EtOAc (100 mL) and then filtered. Filtrate was concentrated andresidue was purified by silica gel chromatography (eluting with 20%-80%ethyl acetate in hexanes) to provide the title compound (3.2 g);MS-(+)-ion: M+1=368.16. In addition,6-(4-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester, was also isolated (5.04 g). MS-(+)-ion: M+1=368.17.

c) 1-Bromo-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

To a solid mixture of7-(4-methoxy-phenoxy)-4-hydroxy-isoquinoline-3-carboxylic acid butylester (305 mg, 0.83 mmol) and N-bromosuccinimide (162 mg, 0.91 mmol)cooled with an ice bath was added acetonitrile (6 mL). Resulting mixturewas stirred at 0° C. for 1.5 h and was concentrated. The residue waspurified by silica gel chromatography (eluting with 10%-40% ethylacetate in hexanes) to provide the title compound (217 mg). ¹H NMR (200MHz, CDCl₃) δ 11.88 (s, 1H), 8.32 (d, J=9.0 Hz, 1H), 7.54 (d, J=2.4 Hz,1H), 7.46 (dd, J=9.0, 2.4 Hz, 1H), 7.08 (d, J=9.4 Hz, 2H), 6.95 (d,J=9.8 Hz, 2H), 4.46 (t, J=7.0 Hz, 2H), 3.85 (s, 3H), 1.85 (m, 2H), 1.47(m, 2H), 0.98 (t, J=7.4 Hz, 3H).

d) 1-Cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-bromo-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (190 mg, 0.43 mmol), copper(I) cyanide (76.3 mg, 0.85 mmol)and N-methyl-pyrrolidine (3 mL) was heated at 130° C. for 1 h. Aftercooled, reaction mixture was partitioned between ethyl acetate andwater. Organic layer was washed with brine, dried over magnesiumsulfate, filtered, and concentrated. Crude product was purified bysilica gel chromatography (eluting with 2%-25% ethyl acetate inmethylene chloride) to give the title compound (129 mg). MS-(+)-ion:M+1=392.80.

e){[1-Cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (110 mg, 0.28 mmol) and glycine (275 mg, 2.82 mmol) in asolution of sodium methoxide (0.5 M in methanol; 5.7 mL) was refluxedovernight. Reaction mixture was concentrated and dissolved in water (50mL). It was washed with ethyl acetate (10 mL). Aqueous layer wasacidified by 1 N HCl to pH=3-4 and extracted with ethyl acetate. Organiclayer was washed with brine, dried over magnesium sulfate, filtered, andconcentrated. Crude product was triturated with methanol (3 mL) andsolid was collected and dried to give the title compound (72 mg).MS-(+)-ion: M+1=394.32.

Example 29[(1-Cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 1-Cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

A mixture of 1-bromo-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (see U.S. Patent Application Publication No.2004/0254215; 350 mg, 0.83 mmol), CuCN (149 mg, 1.66 mmol) andN-methyl-pyrrolidine (2.5 mL) was heated at 130° C. for 2 h. Aftercooled, reaction mixture was poured into water (50 mL) with stirring.Precipitate was collected and rinsed with water. Resulting solid waspartitioned between ethyl acetate and 10% aqueous NH₄OH (50 mL), andvigorously stirred for 15 min. The mixture was acidified by aqueousconcentrated HCl and then 1 N HCl solution to pH=4. The organic phasewas separated and the aqueous phase was extracted with ethyl acetate.Combined organic layers were washed with brine, dried over magnesiumsulfate, filtered and concentrated to give the title product (234 mg).MS-(+)-ion: M+1=369.43.

b)[(1-Cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared from1-Cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carboxylic acid butylester and glycine in analogy to example 28e (94% yield). MS-(+)-ion:M+1=370.32.

Example 30[(6-Benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) 6-Benzenesulfonyl-1-bromo-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

To a mixture of 6-benzenesulfonyl-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (100 mg, 0.26 mmol; see U.S. Patent ApplicationPublication No. 2004/0254215) and benzene (4.5 mL) was added benzoylperoxide (6.3 mg, 0.026 mmol). Resulting mixture was refluxed for 15min. prior to the addition of N-bromosuccinimde (51 mg, 0.29 mmol).Reaction mixture was refluxed overnight. It was concentrated and thecrude product was purified by silica gel chromatography (eluting with20%-80%) ethyl acetate/hexanes to provide the title compound (91 mg).MS-(+)-ion: M+1=466.18, 464.14.

b) 6-Benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester

Prepared from6-benzenesulfonyl-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid butylester and CuCN in analogy to example 29a. Crude product was purified bysilica gel chromatography (2%-20% ethyl acetate in methylene chloride)to give the title compound (49% yield). MS-(+)-ion: M+1=411.30.

c)[(6-Benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

Prepared from6-benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butylester and glycine in analogy to example 28e (56% yield). MS-(+)-ion:M+1=412.26.

Example 31{[1-Cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(4-Benzyloxy-phenoxy)-phthalonitrile

A mixture of 4-benzyloxyphenol (14.53 g, 72.6 mmol),4-nitrophthalonitrile (10.47 g, 60.5 mmol), potassium carbonate (16.69g, 120.9 mmol), and acetone (170 mL) was refluxed overnight; thereaction mixture was cooled, and the solids were filtered off and rinsedwith EtOAc. All liquids were combined and concentrated in vacuo. Theresulting residue was partitioned between EtOAc and 2 M NaOH solution ina 500-mL separation funnel. The organic phase was subsequently washedwith 1 M HCl, saturated NaCl solution, dried over sodium sulfate, andconcentrated in vacuo to give the crude title compound (22.1 g) that wasused in the next step without further purification. ¹H NMR (200 MHz,CDCl₃): δ (ppm)=7.7-6.6 (m, 12H), 5.08 (s, 2H).

b) 4-(4-Benzyloxy-phenoxy)-phthalic acid

A mixture of 4-(4-benzyloxy-phenoxy)-phthalonitrile (22.1 g), KOH (50mL, 45 wt % in water), and MeOH (50 mL) was refluxed for 3 days. Thenwater was added and the resulting solution was acidified to pH 3-4 with6 M HCl. The precipitate formed was collected by filtration, washed withwater, and subsequently dried in vacuo to give the crude title compoundas solid (23.5 g) that was used in the next step without furtherpurification. ¹H NMR (200 MHz, DMSO-d6): δ (ppm)=7.7-6.6 (m, 12H), 5.10(s, 2H).

c) [5-(4-Benzyloxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester and[5-(4-Hydroxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-acetic acidethyl ester

A mixture of 4-(4-Benzyloxy-phenoxy)-phthalic acid (23.5 g, 60.47 mmol)and ethyl glycine HCl salt (8.44 g, 60.47 mmol) was melted with aheating mantle and stirred for 30 min. Then while the mixture was hot,dichloromethane was added to give a solution. After cooling the solutionwas passed through a plug of silica gel. Elution was continued with amixture of EtOAc and dichloromethate (1:1, v/v). The combined fractionswere concentrated in vacuo to give a brown oil, which was purified bycolumn chromatography to give the two title compounds:[5-(4-benzyloxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester (9.41 g) and[5-(4-hydroxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-acetic acidethyl ester (8.6 g).[5-(4-Benzyloxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester: ¹H NMR (200 MHz, CDCl₃): δ (ppm)=7.9-7.2 (m, 12H),5.07 (s, 2H), 4.38 (s, 2H), 4.21 (q, 2H, J=7.2 Hz), 1.27 (t, 3H, J=7.2Hz); [5-(4-Hydroxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester: ¹H NMR (200 MHz, CDCl₃): δ (ppm)=7.9-7.2 (m, 12H),4.40 (s, 2H), 4.21 (q, 2H, J=7.2 Hz), 1.28 (t, 3H, J=7.2 Hz);

d) [1,3-Dioxo-5-(4-propoxy-phenoxy)-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

A mixture of[5-(4-hydroxy-phenoxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-acetic acidethyl ester (2.479 g, 7.26 mmol), 1-bromopropane (1.32 mL, 14.52 mmol),potassium carbonate (2.01 g, 14.52 mmol), and acetone (25 mL) wasrefluxed overnight. After cooling to ambient temperature the mixture wasconcentrated in vacuo and the residue was partitioned between EtOAc andwater. The organic phase was washed with saturated NaCl, dried overanhydrous sodium sulfate, and concentrated in vacuo to give the titlecompound (2.485 g). ¹H NMR (200 MHz, CDCl₃): δ (ppm)=7.77 (q, 1H, J=8.1Hz), 7.3-6.8 (m, 6H), 4.38 (s, 2H), 4.20 (q, 2H, J=7.0 Hz), 3.93 (t, 2H,J=6.4 Hz), 1.82 (q, 2H, 6.6 Hz), 1.28 (t, 3H, J=7.0 Hz), 1.06 (t, 3H,J=7.5 Hz).

e)4-Hydroxy-1-oxo-6-(4-propoxy-phenoxy)-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester

A mixture of[1,3-dioxo-5-(4-propoxy-phenoxy)-1,3-dihydro-isoindol-2-yl]-acetic acidethyl ester (2.485 g, 6.48 mmol), sodium butoxide, and butanol (14.3mmol, 27 mL butanol) was heated at 90° C. to 100° C. for 2 h. Then thereaction mixture was allowed to cool to ambient temperature, acidifiedwith 2 M HCl to pH 3-4, and extracted with EtOAc. The organic phase wasthen washed with water and saturated NaCl solution, dried over anhydroussodium sulfate, and concentrated in vacuo. The residue was purified bycolumn chromatography to give the title compound (722 mg). ESI MS (m/z):412 (M+H)⁺.

f) 1-Bromo-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of4-hydroxy-1-oxo-6-(4-propoxy-phenoxy)-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester (495 mg, 1.20 mmol), POBr₃ (379 mg, 1.32 mmol), andtoluene was microwaved at 110° C. for 25 min. Subsequently, the mixturewas diluted with EtOAc, washed with aqueous NaHCO₃ and saturated NaClsolution before it was dried over anhydrous sodium sulfate andconcentrated in vacuo to give the crude title product (509 mg) that wasused in the next step without further purification. ¹H NMR (200 MHz,CDCl₃): δ (ppm)=11.74 (s, 1H), 8.19 (d, 1H, J=9.2 Hz), 7.60-7.46 (m,2H), 7.07-6.92 (m, 4H), 4.46 (t, 2H, =7.1 Hz), 3.94 (t, 2H, J=6.6 Hz),1.83 (m, 4H), 1.45 (m, 2H), 1.10-0.94 (m, 6H).

g) 1-Cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-bromo-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (315 mg, 0.664 mmol), CuCN (66 mg, 0.730 mmol), andN-methylpyrrolidinone (2 mL) was stirred at 120° C. for 4 h. Aftercooling to room temperature the mixture was poured into EtOAc (20 mL)and saturated ammonium hydroxide solution (˜1 mL) was added. The mixturewas rapidly stirred for 2 min. Then it was acidified with concentratedHCl, washed with water, and saturated NaCl solution before it was driedover anhydrous sodium sulfate, and concentrated in vacuo. The residuewas purified by column chromatography to give title compound (206 mg).ESI MS (m/z): 421 (M+H⁺).

h){[1-Cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carboxylic acidbutyl ester (200 mg, 0.422 mmol), glycine (633 mg, 8.43 mmol) andNaOMe/MeOH solution (12.7 mL, 6.33 mmol) was refluxed overnight. Thenthe mixture was concentrated in vacuo and the residue was dissolved inwater. The solution was acidified with 2 M HCl to pH=3˜4. The resultingprecipitate was collected by filtration, washed with water, andfreeze-dried to give the title compound as a powder (209 mg); ESI MS(m/z): 422 (M+H)⁺.

Example 32{[7-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(Benzo[1,3]dioxol-5-yloxy)-phthalonitrile

Prepared in analogy to example 31a from benzo[1,3]dioxol-5-ol. ¹H NMR(200 MHz, CDCl₃): δ (ppm)=7.8-6.5 (m, 8H).

b) 4-(Benzo[1,3]dioxol-5-yloxy)-phthalic acid

Prepared in analogy to example 31b from4-(benzo[1,3]dioxol-5-yloxy)-phthalonitrile. ¹H NMR (200 MHz, DMSO-d6):δ (ppm)=7.95 (d, 1H, 8.7 Hz), 7.3-6.5 (m, 7 Hz).

c)[5-(Benzo[1,3]dioxol-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

Prepared in analogy to example 31c from4-(benzo[1,3]dioxol-5-yloxy)-phthalic acid. ¹H NMR (200 MHz, CDCl₃): δ(ppm)=7.78 (d, 1H, J=7.4 Hz), 7.29-7.21 (m, 2H), 6.81 (d, 1H, J=6.4 Hz),6.60-6.52 (m, 2H), 4.39 (s, 2H), 4.21 (q, 2H, J=6.8 Hz), 1.28 (t, 3H,J=6.8 Hz).

d)7-(Benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester and6-(Benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31e from[5-(benzo[1,3]dioxol-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester.7-(benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester: ¹H NMR (200 MHz, CDCl₃): δ (ppm)=10.56 (br, s, 1H),8.30 (br, s, 1H), 8.2-6.5 (m, 6H), 6.01 (s, 2H), 4.39 (t, 2H), 1.78 (m,2H), 1.50 (m, 2H), 0.99 (t, 3H, J=7.3 Hz);6-(benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester: ¹H NMR (200 MHz, CDCl₃): δ (ppm)=10.4 (br, s, 1H),8.4-6.5 (m, 7H), 6.02 (s, 2H), 4.40 (t, 2H, J=6.6 Hz), 1.85-1.40 (m,4H), 0.99 (t, 3H, J=7.3 Hz).

e)7-(Benzo[1,3]dioxol-5-yloxy)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31f from7-(benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester and POCl₃. ¹H NMR (200 MHz, CDCl₃): δ (ppm)=11.89 (s,1H), 8.35 (d, 1H, J=9.2 Hz), 7.57 (d, 1H, J=1.9 Hz), 7.50 (m, 1H), 6.83(d, 1H, J=7.8 Hz), 6.65-6.56 (m, 2H), 6.03 (s, 2H), 4.47 (t, 2H, J=7.1Hz), 1.9-1.4 (m, 4H), 0.99 (t, 3H, J=7.3 Hz).

f)7-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of7-(benzo[1,3]dioxol-5-yloxy)-1-chloro-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (118 mg, 0.284 mmol), zinc cyanide (20 mg, 0.17 mmol),zinc (2.2 mg), Pd₂(dba)₃ (13 mg, 0.0142 mmol), dppf (15.7 mg, 0.0284mmol), and dimethylacetamide (1 mL) was stirred at 120° C. for 90 min.After cooling to ambient temperature the reaction mixture was dilutedwith EtOAc, washed with water, and saturated NaCl solution before it wasdried over anhydrous sodium sulfate and concentrated in vacuo. Theresidue was purified by column chromatography to give the title compound(50 mg). ESI MS (m/z): 407 (M+H)⁺.

g){[7-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to example 31h from7-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine. ESI MS (m/z): 406 (M−H)⁻.

Example 33{[6-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a)6-(Benzo[1,3]dioxol-5-yloxy)-1-bromo-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31f from6-(benzo[1,3]dioxol-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester and POBr₃. ESI MS (m/z): 460 (M+H)⁺.

b)6-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31g from6-(benzo[1,3]dioxol-5-yloxy)-1-bromo-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and CuCN. ESI MS (m/z): 407 (M+H)⁺.

c){[6-(Benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to example 31h from6-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine. ESI MS (m/z): 408 (M+H)⁺.

Example 34{[1-Cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 4-(2,3-Dihydro-benzofuran-5-yloxy)-phthalonitrile

Prepared in analogy to example 31a from 2,3-dihydro-benzofuran-5-ol. ¹HNMR (200 MHz, CDCl₃): δ (ppm)=7.7-6.7 (m, 6H), 4.65 (t, 2H, J=7.8 Hz),3.25 (t, 2H, J=7.8 Hz).

b) 4-(2,3-Dihydro-benzofuran-5-yloxy)-phthalic acid

Prepared in analogy to example 31b from4-(2,3-dihydro-benzofuran-5-yloxy)-phthalonitrile. ¹H NMR (200 MHz,DMSO-d6): δ (ppm)=14-13 (br, 2H), 7.78-6.77 (m, 6H), 4.55 (m, 2H), 3.19(m, 2H).

c)[5-(2,3-Dihydro-benzofuran-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

Prepared in analogy to example 31c from4-(2,3-dihydro-benzofuran-5-yloxy)-phthalic acid. ¹H NMR (200 MHz,CDCl₃): δ (ppm)=7.8-6.8 (m, 6H), 4.63 (m, 2H), 4.38 (s, 2H), 4.20 (m,2H), 3.24 (t, 2H, J=4.4 Hz), 1.28 (t, 3H, J=7.1 Hz).

d)7-(2,3-Dihydro-benzofuran-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester and6-(2,3-Dihydro-benzofuran-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31e from[5-(2,3-Dihydro-benzofuran-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester.7-(2,3-Dihydro-benzofuran-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester: ESI MS (m/z) 396 (M+H)⁺;6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester: ESI MS (m/z): 396 (M+H)⁺.

e)1-Bromo-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31f from6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-1-oxo-1,2-dihydro-isoquinoline-3-carboxylicacid butyl ester and POBr₃. ESI MS (m/z): 458 (M+H)⁺.

f)1-Cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

Prepared in analogy to example 31g from1-bromo-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and CuCN. ESI MS (m/z): 405 (M+H)⁺.

g){[1-Cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

Prepared in analogy to example 31h from1-cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester and glycine. ESI MS (m/z): 404 (M−H)⁻.

Example 35[(1-Cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester a)1-Bromo-4-methoxy-8-phenoxy-isoquinoline-3-carboxylic acid methyl ester

A mixture of 1-bromo-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (1.89 g, 5.07 mmol), iodomethane (632 μL, 10.13 mmol),cesium carbonate (3.3 g, 10.13 mmol) and dimethylformamide (15 mL) wasstirred at 45° C. for sixteen hours before it was quenched with water,extracted with ethyl acetate. The organic layer was washed with water,brine, dried over sodium sulfate, concentrated in vacuo and the residuewas purified by flash column chromatography on silica gel with agradient of ethyl acetate and hexanes to give the title compound as ayellow solid (1.03 g): MS: (+) m/z 387.64, 389.75 (M+1, ⁷⁹Br/⁸¹Br).

b) 1-Cyano-4-methoxy-8-phenoxy-isoquinoline-3-carboxylic acid methylester

A mixture of 1-bromo-4-methoxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (1.02 g, 2.63 mmol), copper (I) cyanide (470 mg, 5.25 mmol)and anhydrous dimethylformamide (8.8 mL) was refluxed for ten minutesbefore it was cooled to room temperature quenched with water and ethylacetate. The slurry was filtered. The organic layer was washed withwater, brine, dried over anhydrous sodium sulfate, concentrated in vacuoand the residue was purified by flash column chromatography on silicagel with a gradient of ethyl acetate and hexanes to give the titlecompound as a yellow solid (727 mg): MS: (+) m/z 334.83 (M+1).

c) 1-Cyano-4-methoxy-8-phenoxy-isoquinoline-3-carboxylic acid

To a mixture of 1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carboxylicacid methyl ester (727 mg, 2.18 mmol) and methanol/tetrahydrofuran (12.5mL, 1:1.5) was added 2N sodium hydroxide (5.4 mL, 10.89 mmol) at roomtemperature. The yellow solution was stirred at that temperature forseventy minutes before it was concentrated. Water (20 mL) was added andthe mixture was adjusted to pH=2 with 1N HCl (13 mL). The mixture wasthen extracted with dichloromethane and ethyl acetate. The combinedorganic layers were washed with brine, dried over anhydrous sodiumsulfate, concentrated in vacuo to give the title compound as a yellowsolid (647 mg): MS: (+) m/z 320.80 (M+1).

d) [(1-Cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester

To a mixture of 1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carboxylicacid (483 mg, 1.51 mmol), triethylamine (466 μL, 3.32 mmol), anddichloromethane (12 mL) was added isobutylchloroformate (211 μL, 1.62mmol) at 0° C. The mixture was stirred at 0° C. for twenty-five minutesbefore glycine methyl ester hydrochloride (208 mg, 1.66 mmol) was added.The mixture was stirred at 0° C. for twenty-five minutes and at roomtemperature for 5.5 hours before it was concentrated. The residue waspartitioned between ethyl acetate and 1N HCl. The organic layer waswashed with saturated sodium bicarbonate, brine, dried over anhydroussodium sulfate, concentrated in vacuo and the residue was purified byflash column chromatography on silica gel with a gradient of ethylacetate and hexanes to give the title compound as a yellow solid (343mg): MS: (+) m/z 391.95 (M+1).

Example 36[(1-Cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

To a mixture of[(1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester (21 mg, 0.05 mmol), and methanol/tetrahydrofuran (0.9mL, 1:3.5) was added 2N sodium hydroxide (29.2 μL, 0.06 mmol) at roomtemperature. The yellow solution was stirred at that temperature forfifty-five minutes before it was acidified with 1N HCl (69.1 μL) to pH=3and concentrated in vacuo. The residue was partitioned between ethylacetate and water. The organic layer was dried over anhydrous sodiumsulfate, concentrated in vacuo and the residue was purified by flashcolumn chromatography on silica gel with a gradient of methanol,dichloromethane and acetic acid (0.1%) to give the title compound as apale brown solid (15 mg): MS: (+) m/z 378.00 (M+1).

Example 37(S)-2-[(1-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of 1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (101 mg, 0.31 mmol) and L-Alanine (561 mg, 6.29 mmol) in0.5 M sodium methoxide/methanol (12 mL) was refluxed for four daysbefore it was cooled to room temperature and concentrated in vacuo. Theresidue was dissolved in water (20 mL) and extracted with methyl t-butylether (2×30 mL). The remaining aqueous layer was acidified to pH=3 with1N HCl (8 mL). The resulting white suspension was extracted with ethylacetate (30 mL), dried and concentrated in vacuo to give the titlecompound as a white solid (117 mg): ¹H NMR (DMSO-d₆, 200 MHz): δ=13.52(bs, 1H), 10.11 (br s, 1H), 8.30 (d, 1H), 8.07 (m, 1H), 7.67 (t, J=8 Hz,1H), 7.42 (m, 2H), 7.17 (m, 3H), 4.83 (q, 1H), 1.68 (d, J=7.4 Hz); MS:(+) m/z 378.29 (M+1).

Example 38(R)-2-[(1-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid

A mixture of 1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carboxylic acidmethyl ester (95 mg, 0.30 mmole), D-Alanine (529 mg, 5.94 mmole) and 0.5M sodium methoxide/methanol (11.3 mL) was refluxed for three days beforeit was cooled to room temperature and concentrated in vacuo. The residuewas dissolved in water (25 mL) and extracted with dichloromethane (3×30mL). The remaining aqueous layer was acidified to pH=3 with 1N HCl (8mL). The white suspension was extracted with ethyl acetate (30 mL) anddichloromethane (30 mL). The combined organic layers were washed withbrine, dried over MgSO₄ and concentrated in vacuo to give the titlecompound as a white solid (90 mg): ¹H NMR (DMSO-d₆, 200 MHz): δ=13.52(bs, 1H), 9.15 (br s, 1H), 8.30 (d, 1H), 8.10 (m, 1H), 7.67 (t, J=8 Hz,1H), 7.42 (m, 2H), 7.17 (m, 3H), 4.83 (q, 1H), 1.68 (d, J=7.0 Hz, 3H));MS: (+) m/z 378.29 (M+1).

Example 39{[1-Cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 2-Methyl-benzothiazol-6-ol

A mixture of 6-methoxy-2-methyl-benzothiazole (19.71 g, 0.11 mol),tetrabutylphosphonium bromide (3.73 g, 0.01 mol), and 48% HBr (120 mL)was stirred at 105° C. for twenty-eight hours before it was cooled toroom temperature, neutralized with 10N NaOH (90 mL) and 1N NaOH (45 mL)to pH=4. The resulting precipitate was filtered, washed with water(3×100 mL), dried in vacuo to give the title compound as a pale brownsolid (14.81 g): ¹H NMR (CDCl₃, 200 MHz): δ=7.77 (m, 1H), 7.24 (m, 1H),6.94 (m, 1H), 5.28 (s, 1H), 2.79 (s, 3H).

b) 5-Nitro-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester

A mixture of 4-nitrophthalimide (82.54 g, 0.43 mol), ethyl bromoacetate(78.92 g, 0.47 mol) and potassium carbonate (130.6 g, 0.94 mol), andacetone (1.5 l) was refluxed for eighteen hours before it was cooled toroom temperature, filtered and concentrated in vacuo. The residue waspartitioned between dichloromethane and water. The organic layer wasdried over anhydrous sodium sulfate and concentrated in vacuo to givethe title compound as a yellow solid (107.39 g): MS: (+) m/z 279.25(M+1).

c)[5-(2-Methyl-benzothiazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]aceticacid ethyl ester

A mixture of 2-methyl-benzothiazol-6-ol (2.30 g, 13.92 mmol),5-nitro-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(4.26 g, 15.31 mmol), potassium carbonate (2.3 g, 16.64 mmol) anddimethylacetamide (30 mL) was stirred at 105° C. for twenty hours beforeit was cooled to room temperature and partitioned between ethyl acetateand water. The organic layer was dried over anhydrous sodium sulfate,concentrated in vacuo and the residue was purified by flash columnchromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a yellow solid (2.35 g):MS: (+) m/z 397.19 (M+1).

d)1,4-Dihydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester and1,4-Dihydroxy-7-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

Sodium (1.56 g, 0.07 mol) was dissolved in anhydrous 1-butanol (70 mL)and stirred at 80° C. for one hour. The resulting solution was added toa solution of[5-(2-methyl-benzothiazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester (12.79 g, 0.03 mol) in anhydrous 1-butanol (90 mL). Themixture was stirred at 105° C. for two hours before it was cooled toroom temperature, acidified with 5N HCl (13.5 mL) to pH=5. Theprecipitate was filtered, washed with water, dried in vacuo to give the6,7-isomeric mixture of title compound as a yellow solid (9.9 g): MS:(+) m/z 425.33 (M+1).

e)1-Chloro-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A solution of the regioisomeric mixture of1,4-dihydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butylester/1,4-dihydroxy-7-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester obtained above (3 g, 7.08 mmol), and phosphorusoxychloride (791 μL, 8.49 mmol) in dichloroethane (35 mL) was stirred at120° C. in a CEM microwave apparatus for thirty minutes. The procedurewas repeated three times. The combined reaction mixtures were filtered,saturated sodium bicarbonate solution was added and the mixture wasstirred for one hour. The organic layer was washed with brine, driedover anhydrous sodium sulfate, concentrated in vacuo and the residue waspurified by flash column chromatography on silica gel with a gradient ofethyl acetate and dichloromethane to give a yellow solid (7.05 g). Itwas recrystallized from ethyl acetate (230 mL) to give the titlecompound as a yellow solid (3.42 g): MS: (+) m/z 443.24 (M+1).

f)1-Cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-chloro-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (369 mg, 0.84 mmol),tris(dibenzylideneacetone)dipalladium(0) (38 mg, 0.04 mmol),1,1′-bis(diphenylphosphino)ferrocene (46 mg, 0.08 mmol), zinc cyanide(59 mg, 0.50 mmol), zinc (6 mg, 0.10 mmol) and dimethylacetamide (2 mL)was stirred at 120° C. for two hours and twenty minutes before it wascooled to room temperature, partitioned between ethyl acetate and water,and filtered. The filtrate was washed with water and brine, dried overanhydrous sodium sulfate and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel with a gradient ofethyl acetate and dichloromethane to give the title compound as a whitesolid (245 mg): MS: (+) m/z 434.30 (M+1).

g){[1-Cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (113 mg, 0.26 mmol) and glycine (390 mg, 5.20 mmol) in0.5 M sodium methoxide/methanol (9.9 mL) was refluxed for forty-fivehours before it was cooled to room temperature and concentrated invacuo. The residue was dissolved in water (35 mL) and extracted withmethyl tert-butyl ether (2×25 mL). The remaining aqueous layer wasacidified to pH=3 with 1N HCl (7.4 mL). The white precipitate wasfiltered, washed with water and dried in vacuo to give the titlecompound as a white solid (92 mg): MS: (+) m/z 434.93 (M+1), (−) m/z432.87 (M−1).

Example 40{[1-Cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a) 2-Amino-4-methoxy-phenol

A mixture of 4-methoxy-2-nitrophenol (30.2 g, 0.18 mol), 10% Pd/C,ethanol (200 mL), and ethyl acetate (200 mL) was hydrogenated at ambientpressure and temperature for three days before it was filtered andconcentrated in vacuo to give the title compound as a dark brown solid(20.78 g): ¹H NMR (CDCl₃, 200 MHz): δ=6.64 (m, 1H), 6.32 (m, 1H), 6.21(m, 1H), 3.71 (s, 3H).

b) 5-Methoxy-benzooxazole-2-thiol

A mixture of 2-amino-4-methoxy-phenol (20.7 g, 0.15 mol), potassiumO-ethylxanthate (26.2 g, 0.16 mol) and anhydrous pyridine was refluxedfor two hours before it was cooled to room temperature and ice-cold 1NHCl (600 mL) was added. The precipitate was filtered, washed with waterand dried in vacuo to give the title compound as a pale red solid (22.36g): ¹H NMR (CDCl₃, 200 MHz): δ=7.20 (m, 1H), 6.78 (m, 2H), 3.82 (s, 3H).

c) 2-Chloro-5-methoxy-benzooxazole

To a mixture of 5-methoxy-benzooxazole-2-thiol (12.14 g, 0.07 mol) andthionyl chloride were added two drops of DMF. The mixture was stirred at68° C. for forty minutes and the formation of gas had stopped. Themixture was diluted with dichloromethane, concentrated, and dried invacuo to give the title compound as a green solid (14.31 g): ¹H NMR(CDCl₃, 200 MHz): δ=7.36 (m, 1H), 7.13 (m, 1H), 6.92 (m, 1H), 3.84 (s,3H).

d) (5-Methoxy-benzooxazol-2-yl)-dimethyl-amine

A mixture of 2-chloro-5-methoxy-benzooxazole (1.52 g, 8.31 mmol) and a40% solution of dimethylamine in water (15 mL) was refluxed for twohours before it was cooled to room temperature and partitioned betweenethyl acetate and water. The organic layer was dried over anhydroussodium sulfate and concentrated in vacuo to give the title compound as ablack oil (1.14 g): MS: (+) m/z 397.19 (M+1)

e) 2-Dimethylamino-benzooxazol-5-ol

A mixture of (5-methoxy-benzooxazol-2-yl)-dimethyl-amine (6.41 g, 0.03mol), tetrabutylphosphonium bromide (1.13 g, 3.34 mmol), and 48% HBr (40mL) was stirred at 105° C. for twenty-five hours before it was cooled toroom temperature, neutralized with 10N NaOH (30 mL) to pH=8. Theresulting precipitate was filtered, washed with water, and dried invacuo to give the title compound as a pale brown solid (4.94 g): ¹H NMR(CDCl₃, 200 MHz): δ=7.05 (m, 1H), 6.86 (m, 1H), 6.48 (m, 1H), 3.19 (s,6H).

f)[5-(2-Dimethylamino-benzooxazol-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

A mixture of 2-dimethylamino-benzooxazol-5-ol (4.82 g, 0.03 mmol),5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(8.66 g, 0.03 mmol), potassium carbonate (4.49 g, 0.03 mmol) anddimethylacetamide (30 mL) was stirred at 105° C. for twenty-two hoursbefore it was cooled to room temperature and partitioned betweenchloroform and water. The organic layer was dried over anhydrous sodiumsulfate, concentrated in vacuo and purified by flash columnchromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a orange solid (4.24 g):MS: (+) m/z 410.2 (M+1).

g)6-(2-Dimethylamino-benzooxazol-5-yloxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester and7-(2-Dimethylamino-benzooxazol-5-yloxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester

Sodium (475 mg, 20.65 mmol) was dissolved in anhydrous 1-butanol (24 mL)at 85° C. The resulting freshly prepared solution was added to asolution of[5-(2-dimethylamino-benzooxazol-5-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester (4.23 g, 0.01 mol) in anhydrous 1-butanol (30 mL). Themixture was stirred at 105° C. for one hour and forty-five minutesbefore it was cooled to room temperature, acidified with 2N HCl (10 mL)to pH=5. The precipitate was filtered, washed with water, dried in vacuoto give a regioisomeric mixture of title compounds as a yellow solid(1.2 g): MS: (+) m/z 438.2 (M+1). The filtrate was extracted with ethylacetate. The organic layer was dried over anhydrous sodium sulfate,concentrated in vacuo and the residue was purified by flash columnchromatography on silica gel with a gradient of ethyl acetate anddichloromethane. Fractions corresponding to pure 7-regioisomer werecollected and concentrated to give a yellow solid (452 mg): MS: (+) m/z438.2 (M+1).

h)1-Chloro-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A solution of the regioisomeric mixture of6-(2-dimethylamino-benzooxazol-5-yloxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester and7-(2-dimethylamino-benzooxazol-5-yloxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester obtained as described above (1.05 g, 2.41 mmol), andphosphorus oxychloride (286 μL, 3.13 mmol) in dichloroethane (18 mL) wasstirred at 120° C. in a CEM microwave apparatus for thirty minutes. Themixture was stirred with saturated sodium bicarbonate solution fortwenty minutes. The organic layer was washed with brine, dried overanhydrous sodium sulfate, concentrated in vacuo. Purification by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane gave a solid (463 mg). Recrystallization from ethylacetate (190 mL) furnished the title compound as a yellow solid (219mg): MS: (+) m/z 456.31 (M+1).

i)1-Cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-chloro-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (115 mg, 0.25 mmol),tris(dibenzylideneacetone)dipalladium(0) (12 mg, 0.01 mmol),1,1′-bis(diphenylphosphino)ferrocene (14 mg, 0.02 mmol), zinc cyanide(18 mg, 0.15 mmol), zinc (2 mg, 0.03 mmol) and dimethylacetamide (0.6mL) was stirred at 120° C. for two hours before it was cooled to roomtemperature, partitioned between ethyl acetate and water, filtered. Thefiltrate was washed with water and brine, dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a white solid (53 mg): MS:(+) m/z 447.00 (M+1); (−) m/z 445.20 (M−1).

j){[1-Cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (55 mg, 0.12 mmol), glycine (186 mg, 2.47 mmol) and 0.5M sodium methoxide/methanol (4.7 mL) was refluxed for twenty-five hoursbefore it was cooled to room temperature and concentrated in vacuo. Theresidue was dissolved in water (35 mL) and washed with dichloromethane(2×25 mL). The remaining aqueous layer was acidified to pH=3 with 1N HCl(3.5 mL). The white precipitate was filtered, washed with water anddried in vacuo to give the title compound as a white solid (44 mg): MS:(+) m/z 448.27 (M+1).

Example 41{[1-Cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid a)1-Chloro-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of7-(2-dimethylamino-benzooxazol-5-yloxy)-1,4-dihydroxy-isoquinoline-3-carboxylicacid butyl ester (364 mg, 0.83 mmol), phosphorus oxychloride (92 μL,1.00 mmol) and dichloroethane (4.5 mL) was stirred at 120° C. in a CEMmicrowave apparatus for thirty minutes. The mixture was then stirredwith saturated sodium bicarbonate for fifteen minutes at ambienttemperature. The organic layer was washed with brine, dried overanhydrous sodium sulfate, concentrated in vacuo and the residue waspurified by flash column chromatography on silica gel with a gradient ofethyl acetate and dichloromethane to give the title compound as a whitesolid (220 mg): MS: (+) m/z 456.32 (M+1).

b)1-Cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-chloro-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (92 mg, 0.20 mmol),tris(dibenzylideneacetone)dipalladium(0) (9 mg, 0.01 mmol),1,1′-bis(diphenylphosphino)ferrocene (11 mg, 0.02 mmol), zinc cyanide(14 mg, 0.12 mmol), zinc (2 mg, 0.02 mmol) and dimethylacetamide (0.5mL) was stirred at 120° C. for two hours before it was cooled to roomtemperature, partitioned between ethyl acetate and water, and filtered.The filtrate was washed with water and brine, dried over anhydroussodium sulfate and concentrated in vacuo. The residue was purified byflash column chromatography on silica gel with a gradient of ethylacetate and dichloromethane to give the title compound as a white solid(32 mg): MS: (+) m/z 447.00 (M+1); (−) m/z 445.20 (M−1).

c){[1-Cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carboxylicacid butyl ester (37 mg, 0.08 mmol), glycine (126 mg, 1.68 mmol) and 0.5M sodium methoxide/methanol (3.2 mL) was refluxed for twenty-five hoursbefore it was cooled to room temperature and concentrated in vacuo. Theresidue was dissolved in water (20 mL) and extracted withdichloromethane (2×25 mL). The remaining aqueous layer was acidified topH=3 with 1N HCl (2.4 mL). The white precipitate was filtered, washedwith water and dried in vacuo to give the title compound as a whitesolid (24 mg): MS: (+) m/z 448.27 (M+1).

Example 42{[1-Cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 2-Chloro-6-methoxy-benzothiazole

To a mixture of isoamyl nitrite (17.55 g, 0.15 mol), copper (II)chloride (16.1 g, 0.12 mol) and anhydrous acetonitrile (400 mL) wasadded 2-amino-6-methoxybenzothiazole (18 g, 0.10 mol) over a period offorty minutes at room temperature. The mixture was stirred at 65° C. forthree hours, quenched with 4N HCl (400 mL) and extracted with diethylether (2×300 mL). The organic layer was dried over anhydrous sodiumsulfate, concentrated in vacuo and the residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate andhexanes to give the title compound as a yellow solid (9.97 g): ¹H NMR(CDCl₃, 200 MHz): δ=7.80 (m, 1H), 7.19 (m, 1H), 7.06 (m, 1H), 3.86 (s,3H).

b) 6-Methoxy-2-morpholin-4-yl-benzothiazole

A mixture of 2-chloro-6-methoxy-benzothiazole (9.96 g, 0.05 mol),morpholine (10.86 mL, 0.12 mol) and triethylamine (13.9 mL, 0.10 mol) inethanol (75 mL) was refluxed for twenty-one hours before it was cooledto room temperature and concentrated in vacuo. The residue waspartitioned between dichoromethane and water. The organic layer wasdried over anhydrous sodium sulfate and concentrated in vacuo to givethe title compound as a light yellow solid (12.23 g): MS: (+) m/z 251(M+1)

c) 2-Morpholin-4-yl-benzothiazol-6-ol

A mixture of 6-methoxy-2-morpholin-4-yl-benzothiazole (12.20 g, 48.7mmol), tetrabutylphosphonium bromide (1.65 g, 4.87 mmol) and 48% HBr (80mL) was stirred at 100° C. for twenty hours before it was cooled to roomtemperature, neutralized with 10N NaOH (60 mL) to pH=5-6. The mixturewas extracted with dichloromethane. The organic layer was dried overanhydrous sodium sulfate, concentrated in vacuo to give the titlecompound as a white solid (10.69 g): ¹H NMR (CDCl₃, 200 MHz): δ=7.28 (m,1H), 7.15 (m, 1H), 6.75 (m, 1H), 3.70 (m, 4H), 3.43 (m, 4H).

d)[5-(2-Morpholin-4-yl-benzothiazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester

A mixture of 2-morpholin-4-yl-benzothiazol-6-ol (238 mg, 1.01 mmol),5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(294 mg, 1.06 mmol), potassium carbonate (167 mg, 1.21 mmol) anddimethylacetamide (6 mL) was stirred at 105° C. for twenty-two hoursbefore it was cooled to room temperature and partitioned betweenchloroform and water. The organic layer was dried over anhydrous sodiumsulfate, concentrated in vacuo and the residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a yellow solid (259 mg):MS: (+) m/z 468.21 (M+1).

e)1,4-Dihydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester and1,4-Dihydroxy-7-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

Sodium (703 mg, 30.6 mmol) was dissolved in anhydrous 1-butanol (35 mL)at 85° C. The resulting freshly prepared solution was added to asolution of[5-(2-morpholin-4-yl-benzothiazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester (7.14 g, 15.3 mmol) in anhydrous 1-butanol (55 mL). Themixture was stirred at 110° C. for 90 minutes before it was cooled toroom temperature, acidified with 4N HCl (8 mL) to pH=5. The resultingprecipitate was filtered, washed with water, dried in vacuo to give theregioisomeric mixture of title compound as a yellow solid (6.13 g): MS:(+) m/z 496.19 (M+1).

f)1-Chloro-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A solution of the regioisomeric mixture of1,4-dihydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester and1,4-dihydroxy-7-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester synthesized as described above (1.51 g, 3.04 mmol), andphosphorus oxychloride (417 μL, 4.56 mmol) in dichloroethane (23 mL) wasstirred at 120° C. in a CEM microwave apparatus for thirty minutes. Themixture was then stirred at ambient temperature with saturated sodiumbicarbonate solution for one hour. The organic layer was washed withbrine, dried over anhydrous sodium sulfate, concentrated in vacuo andthe residue was purified by flash column chromatography on silica gelwith a gradient of ethyl acetate and dichloromethane to give a solid(1.05 g). It was recrystallized from ethyl acetate to give the titlecompound as a white solid (484 mg): MS: (+) m/z 514.17 (M+1).

g)1-Cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-Chloro-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (200 mg, 0.39 mmol),tris(dibenzylideneacetone)-dipalladium(0) (18 mg, 0.02 mmol),1,1′-bis(diphenylphosphino)ferrocene (22 mg, 0.04 mmol), zinc cyanide(27 mg, 0.23 mmol), zinc (3 mg, 0.05 mmol) and dimethylacetamide (0.93mL) was stirred at 120° C. for two hours before it was cooled to roomtemperature, partitioned between ethyl acetate and water, filtered. Thefiltrate was washed with water and brine, dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a white solid (137 mg):MS: (+) m/z 505.31 (M+1); (−) m/z 503.31 (M−1).

h){[1-Cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (137 mg, 0.27 mmol), glycine (408 mg, 5.43 mmol) and0.5 M sodium methoxide/methanol (10.3 mL) was refluxed for twenty-sixhours before it was cooled to room temperature and concentrated invacuo. The residue was dissolved in water (40 mL) and extracted withdichloromethane (2×35 mL). The remaining aqueous layer was acidified topH=3 with 1N HCl (7.5 mL). The white precipitate was filtered, washedwith water and dried in vacuo to give the title compound as a whitesolid (119 mg): MS: (+) m/z 506.26 (M+1); (−) m/z 504.24 (M−1).

Example 43{[1-Cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid a) 1-(2,4-Dihydroxy-phenyl)-ethanone oxime

A mixture of 2,4-dihydroxyacetophenone (25 g, 0.16 mol), hydroxylaminehydrochloride (14.8 g, 0.21 mol), sodium acetate (20 g, 0.24 mol) andwater/dioxane (300 mL, 1:1) was stirred at room temperature for threedays before the mixture was partitioned between chloroform and water.The organic layer was dried over anhydrous sodium sulfate, concentratedin vacuo and the residue was purified by flash column chromatography onsilica gel with a gradient of ethyl acetate and dichloromethane to givethe title compound as a white solid (11.67 g): ¹H NMR (CD₃OD, 200 MHz):δ=7.26 (m, 1H), 6.28 (m, 2H), 4.88 (s, 2H), 3.29 (m, 1H), 2.24 (s, 3H).

b) 2-Methyl-benzooxazol-6-ol

To a mixture of 1-(2,4-dihydroxy-phenyl)-ethanone oxime (4.2 g, 25 mmol)and acetonitrile/dimethylacetamide (20 mL, 3:1) cooled by a water bath(20-25° C.) was added dropwide phosphorus chloride (2.4 mL, 26.2 mmol)over a period of fifteen minutes. The mixture was stirred for anadditional thirty minutes before it was poured into ice water containingsodium acetate (6 g). The mixture was stirred for ten minutes, filtered,and the filter cake was washed with water and dried in vacuo to give thetitle compound as a yellow solid (3.15 g): ¹H NMR (CDCl₃, 200 MHz):δ=7.45 (m, 1H), 6.96 (m, 1H), 6.80 (m, 1H), 2.60 (s, 3H).

c)[5-(2-Methyl-benzooxazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]aceticacid ethyl ester

A mixture of 2-methyl-benzooxazol-6-ol (8.41 g, 56 mmol),5-nitro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetic acid ethyl ester(17.23 g, 62 mmol), potassium carbonate (9.36 g, 67.7 mmol), anddimethylacetamide (120 mL) was stirred at 105° C. for twenty hoursbefore it was cooled to room temperature and partitioned betweenchloroform and water. The organic layer was dried over anhydrous sodiumsulfate, concentrated in vacuo and the residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a yellow solid (9.18 g):MS: (+) m/z 381.27 (M+1).

d)1,4-Dihydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester and1,4-Dihydroxy-7-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

Sodium (1.11 g, 48.2 mmol) was dissolved in anhydrous 1-butanol (60 mL)at 90° C. The resulting freshly prepared solution was added to asolution of[5-(2-methyl-benzooxazol-6-yloxy)-1,3-dioxo-1,3-dihydro-isoindol-2-yl]-aceticacid ethyl ester (9.16 g, 24.1 mmol) in anhydrous 1-butanol (70 mL). Themixture was stirred at 110° C. for two hours before it was cooled toroom temperature, acidified with 2N HCl (24 mL) to pH=4. The precipitatewas filtered, washed with water, dried in vacuo to give the 6,7-isomericmixture of title compound as a yellow solid (6.63 g): MS: (+) m/z 409.34(M+1).

e)1-Chloro-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A solution of the regiosomeric mixture of1,4-dihydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester and1,4-dihydroxy-7-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester synthesized as described above (2.5 g, 6.12 mmol), andphosphorus oxychloride (685 μL, 7.35 mmol) in dichloroethane (30 mL) wasstirred at 120° C. in a CEM microwave apparatus for thirty minutes. Themixture was stirred at ambient temperature with a saturated sodiumbicarbonate solution for thirty minutes. The organic layer was washedwith brine, dried over anhydrous sodium sulfate, concentrated in vacuoand the residue was purified by flash column chromatography on silicagel with a gradient of ethyl acetate and dichloromethane to give a solid(1.72 g). It was recrystallized from ethyl acetate (130 mL) to give thetitle compound as a white solid (844 mg): MS: (+) m/z 427.16 (M+1).

f)1-Cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester

A mixture of1-chloro-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (199 mg, 0.47 mmol),tris(dibenzylideneacetone)dipalladium(0) (21 mg, 0.02 mmol),1,1′-bis(diphenylphosphino)ferrocene (26 mg, 0.04 mmol), zinc cyanide(33 mg, 0.28 mmol), zinc (4 mg, 0.06 mmol) and dimethylacetamide (1.1mL) was stirred at 120° C. for two hours before it was cooled to roomtemperature, partitioned between ethyl acetate and water, filtered. Thefiltrate was washed with water and brine, dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography on silica gel with a gradient of ethyl acetate anddichloromethane to give the title compound as a white solid (80 mg): MS:(+) m/z 418.38 (M+1).

g){[1-Cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid

A mixture of1-cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carboxylicacid butyl ester (81 mg, 0.19 mmol), glycine (292 mg, 3.89 mmol), and0.5 M sodium methoxide/methanol (7.4 mL) was refluxed for twenty-sixhours before it was cooled to room temperature and concentrated invacuo. The residue was dissolved in water (25 mL) and extracted withdichloromethane (2×35 mL). The remaining aqueous layer was acidified topH=3 with 1N HCl (5.5 mL). The precipitate was filtered, washed withwater and dried in vacuo to give the title compound as a brown solid (72mg): MS: (+) m/z 419.30 (M+1); (−) m/z 417.28 (M−1).

Example 44[(6-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acida) 6-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid butyl ester

A mixture of 1-bromo-6-chloro-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (509 mg, 1.43 mmol; prepared according to US 2004/0254215A1), copper (I) cyanide (255 mg, 2.85 mmol) and anhydrousdimethylformamide (6 mL) was refluxed for twenty-five minutes before itwas cooled to room temperature and quenched with water.Chloroform/2-propanol (75 mL, 3:1) was added and the mixture was stirredfor ten minutes before it was filtered. The organic layer was washedwith water, brine, dried over anhydrous sodium sulfate, concentrated invacuo and the residue was purified by flash column chromatography onsilica gel with a gradient of ethyl acetate and dichloromethane to givethe title compound as a yellow solid (280 mg): MS: (−) m/z 303.20 (M−1).

b) [(6-Chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

A mixture of 6-chloro-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acidbutyl ester (99 mg, 0.33 mmol) and glycine (1.84 g, 24.49 mmol) in 0.5 Msodium methoxide/methanol (32.6 mL) was refluxed for twenty-one hoursbefore it was cooled to room temperature and concentrated in vacuo. Theresidue was dissolved in water (40 mL) and the solution washed withdichloromethane (3×50 mL). The remaining aqueous layer was acidified topH=3 with 1N HCl. The suspension was extracted with ethyl acetate (50mL). The organic layer was dried over anhydrous sodium sulfate andconcentrated in vacuo to give the title compound as a white solid (84mg): MS: (+) m/z 306.27 (M+1); (−) m/z 304.26 (M−1).

Example 45[(7-Butoxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

[(7-Butoxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acidwas prepared by adding 135 mg (0.383 mmol) of[(7-butoxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic(prepared according to U.S. Pat. No. 6,093,730), 18 mg (0.02 mmol)tris(dibenzylideneacetone)dipalladium(0), 22.2 mg (0.04 mmol)1,1′-bis(diphenylphosphino) ferrocene, 3 mg (0.04 mmol) zinc dust, and27 mg (0.23 mmol) zinc cyanide to 0.80 mL of N,N-dimethylacetamide. Theresultant mixture was heated at 115° C. for 3 hours under a nitrogenatmosphere, and then cooled to room temperature. The mixture was dilutedwith ethyl acetate and extracted three times with aqueous sodiumbicarbonate solution. The aqueous extracts were acidified withconcentrated HCl, and the resultant white precipitate was collected: 28mg of the title compound; MS (ESI−): m/z 342.0 (M−1)

Example 46[(1-Cyano-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

[(1-Cyano-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid was prepared by adding 76 mg (0.164 mmol) of[(1-chloro-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to US 2006/0217416), 7.3 mg (0.008 mmol)tris(dibenzylideneacetone)dipalladium(0), 9.0 mg (0.016 mmol)1,1′-bis(diphenylphosphino) ferrocene, 1.3 mg (0.020 mmol) zinc dust,and 12 mg (0.10 mmol) zinc cyanide to 0.35 mL of N,N-dimethylacetamide.The resultant mixture was heated at 115° C. for 3 hours under a nitrogenatmosphere, and then cooled to room temperature. The mixture was dilutedwith ethyl acetate and extracted three times with aqueous sodiumbicarbonate and twice with 1 N NaOH solutions. The basic aqueousextracts were acidified with concentrated HCl, and extracted three timeswith ethyl acetate. The organic extracts were dried over sodium sulfateand concentrated to give 29 mg of the title compound as a white solid;MS (ESI−): m/z 454.0 (M−1)

Example 47[(1-Cyano-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

[(1-Cyano-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid was prepared by adding 159 mg (0.511 mmol) of[(1-chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to U.S. Pat. No. 6,093,730), 24 mg (0.025 mmol)tris(dibenzylideneacetone)dipalladium(0), 28 mg (0.051 mmol)1,1′-bis(diphenylphosphino) ferrocene, 4 mg (0.06 mmol) zinc dust, and36 mg (0.306 mmol) zinc cyanide to 1.0 mL of N,N-dimethylacetamide. Theresultant mixture was heated at 115° C. for 3 hours under a nitrogenatmosphere, and then cooled to room temperature. The mixture was dilutedwith ethyl acetate and half-saturated aqueous sodium bicarbonatesolution and filtered through a celite pad. The biphasic mixture wasseparated, and the basic aqueous extract was acidified with concentratedHCl and extracted three times with ethyl acetate. The organic extractswere dried over sodium sulfate and concentrated to give 107 mg of thetitle compound as a white solid; MS (ESI+): m/z 302.3 (M+1)

Example 48[(1-Cyano-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

[(1-Cyano-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid was prepared from[(1-chloro-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to U.S. Pat. No. 6,093,730) under conditionsanalogous to example 47a; MS (ESI+): m/z 330.3 (M+1)

Example 49[(1-Cyano-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid

[(1-Cyano-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid was prepared from[(1-chloro-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to US2006/217416) under conditions analogous toexample 47a; MS (ESI+): m/z 330.3 (M+1)

Example 50[(1-Cyano-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

[(1-Cyano-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acidwas prepared from[(1-chloro-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to US2006/217416) under conditions analogous toexample 47a; MS (ESI+): m/z 348.3 (M+1)

Example 51[(1-Cyano-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acid

[(1-Cyano-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-acetic acidwas prepared from[(1-Chloro-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid (prepared according to US2006/217416) under conditions analogous toexample 47a; MS (ESI+): m/z 348.3 (M+1)

Example 52[(7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid a) (2,4-Dimethoxy-benzylamino)-acetic acid ethyl ester

A mixture of 2,4-dimethoxy-benzaldehyde (50 g, 0.30 mole), glycine ethylester hydrochloride (44 g, 0.32 mole) and triethylamine (43.9 mL, 0.32mole) in dichloroethane was stirred for one hour before sodiumtriacetoxyborohydride (100 g, 0.47 mole) was added in four portions. Themixture was stirred for three days before it was quenched with saturatedsodium bicarbonate. The organic layer was extracted with 3N HCl (2×400mL, 2.4 mole). The aqueous layers were combined, adjusted with solidsodium hydroxide (97.68 g, 2.4 mole) to pH=8 and extracted with ethylacetate (500 mL). The ethyl acetate extract was washed with brine, driedover sodium sulfate, and concentrated. The residue was purified by flashcolumn chromatography with a gradient of methanol and dichloromethane onsilica gel (120 g) to give the title compound as a yellow oil (42.77 g)¹H NMR (CDCl₃, 200 MHz): δ=7.11 (m, 1H), 6.41 (m, 2H), 4.15 (q, J=7.2Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.74 (s, 2H), 3.37 (s, 2H), 1.27(t, J=7.2 Hz, 3H).

b) 4-(tert-Butyl-dimethyl-silanyloxy)-2-methyl-benzoic acid ethyl ester

A mixture of 18 g (100 mmol) of 4-hydroxy-2-methyl-benzoic acid ethylester (prepared according to Sen, et. al. (1987), Indian J. Chem. Sec B,26: 679-682), 10.2 g (150 mmol) of imidazole, and 17.2 g (115 mmol) oftert-butyldimethylsilyl chloride in 110 mL of anhydrous DMF was stirredfor 24 hours at room temperature. The reaction was diluted with waterand extracted with ethyl acetate. The organic fraction was washed withwater, 0.1 N HCl, and brine, then dried over anhydrous magnesium sulfateand concentrated to 28.8 g of the title compound; MS (ESI+): m/z 295.5(M+1)

c) N-(2,4-dimethoxy-benzyl),N-(2-ethoxycarbonyl-5-hydroxy-benzyl)glycineethyl ester

A mixture of 20 g (68 mmol) of4-(tert-butyl-dimethyl-silanyloxy)-2-methyl-benzoic acid ethyl ester,12.1 g (68 mmol) of N-bromosuccinimide, 1.5 g (6.8 mmol) of benzoylperoxide in 230 mL of carbon tetrachloride was heated at refluxtemperature for 5 hours. The resultant mixture was cooled, filteredthrough a plug of silica gel, and concentrated under reduced pressure.To the residue (12.2 g) was added 8.0 g (28.7 mmol) ofN-(2,4-dimethoxy-benzyl)glycine ethyl ester, 3.96 g (28.7 mmol) ofpotassium carbonate, and 65 mL of anhydrous DMF and the mixture wasstirred for 6.5 hours. The mixture was then partitioned into a biphasicwater-ethyl acetate mixture and the isolated organic layer was washedwith brine, dried over anhydrous magnesium sulfate, and concentrated.The residue was purified by flash chromatography: eluting the desiredproduct from silica gel with a gradient of 10-90% ethyl acteate inhexanes. 5.33 g of the title compound (yellow oil) was isolated; MS(ESI+): 432.2 (M+1)

d)N-(2,4-dimethoxy-benzyl),N-(5-benzyloxy-2-ethoxycarbonyl-benzyl)glycineethyl ester

A solution of 5.3 g (12.3 mmol) ofN-(2,4-dimethoxy-benzyl),N-(2-ethoxycarbonyl-5-hydroxy-benzyl)glycineethyl ester, 1.8 mL (15.4 mmol) benzylbromide, and 4.4 g (13.5 mmol)cesium carbonate in 35 mL of anhydrous DMF was stirred for 18 hours. Thereaction mixture was partitioned between diethyl ether and water, andthe organic fraction washed with saturated aqueous sodium bicarbonate,brine, dried over anhydrous sodium sulfate, filtered through a pad ofsilica gel, and concentrated under reduced pressure to 6.4 g of thetitle compound. MS (ESI+) 522.5 e/z (M+1)

e)7-Benzyloxy-2-(2,4-dimethoxy-benzyl)-4-hydroxy-1,2-dihydro-isoquinoline-3-carboxylicacid ethyl ester

A solution of 6.4 g (12.3 mmol) ofN-(2,4-dimethoxy-benzyl),N-(5-benzyloxy-2-ethoxycarbonyl-benzyl)glycineethyl ester in 140 mL of anhydrous THF was cooled in an ice bath. 24.6mL of 1 N potassium tert-butoxide in THF was added slowly to thestirring cold solution. The reaction mixture was stirred at 0° C. for 1hour and then allowed to warm to room temperature and stirred for 4hours. The mixture was poured into a biphasic mixture of ethyl acetateand saturated aqueous ammonium chloride. The organic fraction was washedtwice with brine, dried over anhydrous sodium sulfate, and concentratedunder reduced pressure to give 5.7 g of the title compound; MS (ESI+):476.3 e/z (M+1)

f) 7-Benzyloxy-4-hydroxy-isoquinoline-3-carboxylic acid ethyl ester

Dichloromethane (100 mL) was cooled to 0° C. and 5.7 g (12 mmol) of7-benzyloxy-2-(2,4-dimethoxy-benzyl)-4-hydroxy-1,2-dihydro-isoquinoline-3-carboxylicacid ethyl ester and 1.31 mL (18 mmol) of thionyl chloride were added.The reaction was stirred for one hour and then allowed to warm to roomtemperature and stirred for an additional 3 hours. 60 mL of hexanes wasadded to the resultant slurry and the white solid was collected byfiltration through a medium glass fitted filter. The solid waspartitioned between ethyl acetate and saturated sodium bicarbonatesolution and the organic phase was washed with brine, dried overanhydrous sodium sulfate, and concentrated to give the title compound(2.7 g, white solid); MS (ESI+): 324.3 e/z (M+1)

g) 7-Benzyloxy-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid ethylester

A suspension of 2.48 g (7.67 mmol) of7-benzyloxy-4-hydroxy-isoquinoline-3-carboxylic acid ethyl ester and1.43 g (8.0 mmol) of N-bromosuccinimide in 25 mL of acetonitrile washeated to 70° C. for ten minutes. The reaction was cooled and a whitesolid precipitated out of solution. The solid was collected byfiltration through a medium glass fritted filter and washed with coldacetonitrile to give 2.3 g of the title compound; MS (ESI+): 402.2,404.2 e/z (M+1, ⁷⁹Br/⁸¹Br)

h) 7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid ethylester

7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid ethyl esterwas prepared by heating a mixture of 1.76 g (4.4 mmol) of7-benzyloxy-1-bromo-4-hydroxy-isoquinoline-3-carboxylic acid ethylester, 201 mg (0.2.0 mmol) tris(dibenzylideneacetone)dipalladium(0), 243mg (0.44 mmol) 1,1′-bis(diphenylphosphino) ferrocene, 34 mg (0.53 mmol)zinc dust, 309 mg (2.64 mmol) zinc cyanide, and 9.0 mL ofN,N-dimethylacetamide at 115 deg C. for 3 hours. The resultant mixturewas diluted with ethyl acetate and saturated aqueous ammonium chlorideand filtered through a celite pad. The organic fraction was washed withsaturated ammonium chloride, water, brine, dried over anhydrous sodiumsulfate, and concentrated in vacuo. The residue was purified by flashchromatography, eluting from silica gel with a 10 to 90 percent gradientof ethyl acetate in hexanes to give 1.18 g of the title compound; MS(ESI+): 349.3 e/z (M+1)

i)[(7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid

[(7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid was prepared by heating a solution of 110 mg (0.316 mmol) of7-benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid ethyl esterand 210 mg (2.8 mmol) of glycine in 5 mL of 0.5 N NaOMe in methanol atreflux temperature for 28 hours. The reaction mixture was cooled andacidified with 3 mL of 1 N HCl. A white solid was collected on a mediumglass fritted filter to provide 115 mg of the title compound; MS (ESI+):378.2 e/z (M+1)

Example 53[(1-Cyano-4,7-dihydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid a)1-Cyano-4,7-dihydroxy-isoquinoline-3-carboxylic acid ethyl ester

1-Cyano-4,7-dihydroxy-isoquinoline-3-carboxylic acid ethyl ester wasprepared by heating a mixture of 175 mg (0.50 mmol) of7-Benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carboxylic acid ethyl ester(example 52h), 630 mg (10 mmol) of ammonium formate, and 40 mg of 10%Pd/C in 3 mL of 1:1 EtOAc and EtOH at reflux temperature for 40 min. Theresultant mixture was cooled, filtered through a celite pad to removesolids, and concentrated to a crude solid. The solid was triturated withhot ethanol to give 56 mg of off-white solid upon drying; MS (ESI+):259.3 e/z (M+1)

b) [(1-Cyano-4,7-dihydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid

[(1-Cyano-4,7-dihydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid wasprepared by heating a mixture of 42 mg (0.162 mmol) of 1-Cyano-4,7dihydroxy-isoquinoline-3-carboxylic acid ethyl ester and 97 mg (1.29mmol) of glycine in 2.25 mL of 0.5 N NaOMe in methanol at refluxtemperature for 26 hours. The reaction mixture was cooled and acidifiedwith 3 mL of 1 N HCl and water. A white solid was collected on a mediumglass fritted filter to provide 40 mg of the title compound; MS (ESI+):288.2 e/z (M+1)

Example 54 Comparative Assay of Compounds with Chloro, Bromo, Hydrogen,or Methyl in Place of Cyano at C-1 Position

Compounds were tested for activity using the following assay. Compoundswere dissolved in aqueous solution containing twice as much sodiumhydroxide, mole-for-mole, as compound and dextrose as tonicity agent.Male Swiss Webster mice were dosed by tail vein injection with thecompound, and blood samples were collected into EDTA and heparin 4 hourspost IV dosing. The samples were analyzed using a mouse erythropoietinQUANTIKINE ELISA kit (R&D Systems Inc., Minneapolis Minn.) according tothe manufacturer's instructions. Compounds of the present inventionshowed measurable increase in plasma erythropoietin levels. Further,compounds containing the cyano substitution at C-1, as presentlyclaimed, surprisingly produce higher, e.g., at least two times higher,plasma erythropoietin levels than a comparable compound containing ahydrogen, chloro, bromo, or methyl at C-1 position at a given dose.Table 1 below illustrates the fold difference (i.e., the level ofimprovement) between erythropoietin levels achieved by compounds of theinvention compared to erythropoietin levels achieved by compounds havingthe same structure except the cyano group at the C-1 position isreplaced as shown below, tested under the same conditions and at thesame concentration.

TABLE 1 Comparative Fold Example Substituent Difference 1 —Cl  4.7× 3—CH₃  2.5× 6 —Cl   31× 9 —CH₃  2.4× 12 —Cl  449× 12 —Br  362× 39 —CH₃ 178× 40 —H   81× 20 —H   23× 22 —H   26× 23 —H  277×

1.-46. (canceled)
 47. A method of treating anemia, the method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising one or more compoundsrepresented by formula I:

wherein: R is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl; R¹, R², R³ and R⁴ are independently selected from thegroup consisting of hydrogen, halo, cyano, hydroxyl, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, —OR⁷, —SR⁷, —SOR⁷, and —SO₇R⁷ wherein R⁷ isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl; R⁵ and R⁶ are independently selected fromthe group consisting of hydrogen or C₁₋₃ alkyl; or a pharmaceuticallyacceptable salt, tautomer, or stereoisomer thereof; or an ester or amideof the carboxylic acid moiety on the glycine or alanine-basedsubstituent of the cyanoisoquinoline ring, and a pharmaceuticallyacceptable excipient.
 48. The method of claim 47, wherein at least twoof R¹, R², R³ and R⁴ are hydrogen.
 49. The method of claim 47, whereinR¹, R², R³, and R⁴ are independently selected from the group consistingof hydrogen, hydroxyl, halo, substituted alkyl, aryl, —OR⁷, —SR⁷, and—SO₂R⁷ wherein R⁷ is selected from the group consisting of alkyl,substituted alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl.
 50. The method of claim 47, wherein R¹, R², R³,and R⁴ are selected from the group consisting of hydrogen, halo,haloalkyl, alkyl, alkoxy, aryloxy and substituted aryloxy.
 51. Themethod of claim 47, wherein R¹, R², R³, and R⁴ are independentlyselected from the group consisting of hydrogen, hydroxyl, phenyl,chloro, trifluoromethyl, benzyl, benzyloxy, methoxy, butoxy, isopropoxy,phenoxy, 4-fluorophenoxy, 2-methoxyphenoxy, 3-methoxyphenoxy,4-methoxyphenoxy, 2,6-dimethylphenoxy, 2-ethyl-6-methylphenoxy,2,4,6-trimethylphenoxy, 4-chloro-2,6-dimethylphenoxy, 4-propoxyphenoxy,2,3-dihydro-benzofuran-5-yloxy, 2-methyl-benzothiazol-6-yloxy,2-dimethylamino-benzooxazol-5-yloxy,2-morpholin-4-yl-benzothiazol-6-yloxy, 2-methyl-benzooxazol-6-yloxy,benzo[1,3]dioxo-5-yloxy, phenylsulfanyl, phenylsulfonyl, andcyclohexyloxy.
 52. The method of claim 51, wherein R¹, R², R³, and R⁴are selected from the group consisting of hydrogen, chloro, methoxy,trifluoromethyl, phenoxy, and 4-fluorophenoxy.
 53. The method of claim51, wherein R¹ and R⁴ are hydrogen.
 54. The method of claim 51, whereinR¹, R³ and R⁴ are hydrogen.
 55. The method of claim 51, wherein R¹, R²and R³ are hydrogen.
 56. The method of claim 51, wherein R¹, R², and R⁴are hydrogen.
 57. The method of claim 51, wherein R², R³, and R⁴ arehydrogen.
 58. The method of claim 51, wherein R¹, R², R³, and R⁴ arehydrogen.
 59. The method of claim 51, where R¹ is selected from thegroup consisting of phenyl, phenoxy and 4-fluorophenoxy.
 60. The methodof claim 47, wherein R² is selected from the group consisting of halo,cyano, hydroxyl, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino, —OR⁷,—SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷ is selected from the group consistingof alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl.
 61. The methodof claim 60, wherein R² is selected from the group consisting of halo,—OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷ is selected from the groupconsisting of alkyl, substituted alkyl, cycloalkyl, aryl, substitutedaryl, heteroaryl, and substituted heteroaryl.
 62. The method of claim60, wherein R² is selected from the group consisting of chloro, methoxy,isopropoxy, phenoxy, 4-fluorophenoxy, 4-methoxyphenoxy,2,6-dimethylphenoxy, 2-ethyl-6-methylphenoxy, 2,4,6-trimethylphenoxy,4-chloro-2,6-dimethylphenoxy, 4-propoxyphenoxy,2,3-dihydro-benzofuran-5-yloxy, 2-methyl-benzothiazol-6-yloxy,2-dimethylamino-benzooxazol-5-yloxy,2-morpholin-4-yl-benzothiazol-6-yloxy, 2-methyl-benzooxazol-6-yloxy,benzo[1,3]dioxo-5-yloxy, phenylsulfonyl, phenylsulfanyl, andcyclohexyloxy.
 63. The method of claim 60, wherein R² is methoxy,phenoxy, or 4-fluorophenoxy.
 64. The method of claim 47, wherein R³ isselected from the group consisting of hydroxyl, halo, haloalkyl,substituted alkyl, —OR⁷, —SR⁷, —SOR⁷, and —SO₂R⁷ wherein R⁷ is selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, and substituted heteroaryl.
 65. The method of claim64, wherein R³ is selected from the group consisting of trifluoromethyl,chloro, hydroxyl, benzyl, methoxy, isopropoxy, butoxy, benzyloxy,phenoxy, 4-fluorophenoxy, 2,6-dimethylphenoxy, 4-methoxyphenoxy,2-dimethylamino-benzooxazol-5-yloxy, benzo[1,3]dioxo-5-yloxy, andphenylsulfanyl.
 66. The method of claim 64, wherein R³ is phenoxy,4-fluorophenoxy, trifluoromethyl, or chloro.
 67. The method of claim 47,wherein R⁴ is selected from the group consisting of phenyl, phenoxy,2-methoxyphenoxy, 3-methoxyphenoxy, 4-methoxyphenoxy, and4-fluorophenoxy.
 68. The method of claim 67, wherein R⁴ is phenoxy or4-fluorophenoxy.
 69. The method of claim 47, wherein R⁵ is hydrogen. 70.The method of claim 47, wherein R⁵ is methyl.
 71. The method of claim47, wherein R⁶ is hydrogen.
 72. The method of claim 47, wherein R⁶ ismethyl.
 73. The method of claim 47, wherein R is hydrogen.
 74. Themethod of claim 47, wherein R is methyl.
 75. The method of claim 47,wherein the compound is of formula II:

wherein: R³¹, R³², R³³ and R³⁴ are independently selected from the groupconsisting of hydrogen, cyano, hydroxyl, halo, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino,substituted amino, —OR³⁷, —SR³⁷, —SOR³⁷, and —SO₂R³⁷ wherein R³⁷ isselected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,and substituted heteroaryl; R³⁵ is hydrogen or methyl; or apharmaceutically acceptable salt, tautomer, or stereoisomer thereof; oran ester or amide of the carboxylic acid moiety on the glycine- oralanine-based substituent of the cyanoisoquinoline ring.
 76. The methodof claim 75, wherein at least three of R³¹, R³², R³³, and R³⁴ arehydrogen.
 77. The method of claim 75, wherein R³¹, R³², R³³ and R³⁴ areindependently selected from the group consisting of hydrogen, halo,hydroxy, alkyl, substituted alkyl, haloalkyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy,heteroaryloxy, substituted heteroaryloxy, cycloalkoxy, substitutedcycloalkoxy, amino, and substituted amino; and R³⁵ is hydrogen ormethyl.
 78. The method of claim 75, wherein R³¹, R³², R³³ and R³⁴ areindependently selected from the group consisting of hydrogen,substituted alkyl, aryl, aryloxy, and substituted aryloxy; and R³⁵ ishydrogen or methyl.
 79. The method of claim 47, wherein the compound isselected from the group consisting of:{[1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-acetic acid,2-(S)-[(1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl]-amino}-aceticacid,2-(S)-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,2-(R)-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-7-(4-fluorophenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-(trifluoromethyl)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-chloro-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-8-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-6-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-6-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-6-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-hydroxy-5-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-4-hydroxy-8-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-(3-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-8-(2-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(7-benzyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-5-(4-fluoro-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-7-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2-ethyl-6-methyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2,4,6-trimethyl-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[6-(4-chloro-2,6-dimethyl-phenoxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-6-cyclohexyloxy-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(6-benzenesulfonyl-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,{[1-cyano-4-hydroxy-6-(4-propoxy-phenoxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[7-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[6-(benzo[1,3]dioxol-5-yloxy)-1-cyano-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2,3-dihydro-benzofuran-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,[(1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid methyl ester,[(1-cyano-4-methoxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,(S)-2-[(1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,(R)-2-[(1-cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionicacid,{[1-cyano-4-hydroxy-6-(2-methyl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-6-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-7-(2-dimethylamino-benzooxazol-5-yloxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2-morpholin-4-yl-benzothiazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,{[1-cyano-4-hydroxy-6-(2-methyl-benzooxazol-6-yloxy)-isoquinoline-3-carbonyl]-amino}-aceticacid,[(6-chloro-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(7-butoxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6,7-diphenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-7-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-6-isopropoxy-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-5-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(1-cyano-4-hydroxy-8-phenyl-isoquinoline-3-carbonyl)-amino]-aceticacid,[(7-benzyloxy-1-cyano-4-hydroxy-isoquinoline-3-carbonyl)-amino]-aceticacid, and [(1-cyano-4,7-dihydroxy-isoquinoline-3-carbonyl)-amino]aceticacid, or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof; or an ester or amide of the carboxylic acid moiety on theglycine- or alanine-based substituent of the cyanoisoquinoline ring.